RNA INTERFERENCE MEDIATED TREATMENT OF ALZHEIMER&#39;S DISEASE USING SHORT INTERFERING NUCLEIC ACID (siNA)

ABSTRACT

The present invention concerns methods and reagents useful in modulating BACE gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against beta-secretase (BACE), amyloid precursor protein (APP), pin-1, presenillin 1 (PS-1) and/or presenillin 2 (PS-2) gene expression and/or activity. The small nucleic acid molecules are useful in the treatment of Alzheimer&#39;s disease and any other condition that responds to modulation of BACE, APP, pin-1, PS-1 and/or PS-2 expression or activity.

This application is a continuation of U.S. patent application Ser. No.10/607,933, filed Jun. 27, 2003, which is a continuation-in-part of U.S.patent application Ser. No. 10/444,853, filed May 23, 2003, which is acontinuation-in-part of International Patent Application No.PCT/US03/05346, filed Feb. 20, 2003, and a continuation-in-part ofInternational Patent Application No. PCT/US03/05028, filed Feb. 20,2003, and a continuation-in-part of U.S. patent application Ser. No.09/930,423, filed Aug. 15, 2001 and a continuation-in-part ofInternational Patent Application No. PCT/US03/04710, filed Feb. 18,2003, which is a continuation-in-part of U.S. patent application Ser.No. 10/205,309, filed Jul. 25, 2002, and claims the benefit of U.S.Provisional Application No. 60/358,580, filed Feb. 20, 2002, U.S.Provisional Application No. 60/363,124, filed Mar. 11, 2002, U.S.Provisional Application No. 60/386,782, filed Jun. 6, 2002, U.S.Provisional Application No. 60/406,784, filed Aug. 29, 2002, U.S.Provisional Application No. 60/408,378, filed Sep. 5, 2002, U.S.Provisional Application No. 60/409,293, filed Sep. 9, 2002, and U.S.Provisional Application No. 60/440,129, filed Jan. 15, 2003. The instantapplication claims priority to all of the listed applications, which arehereby incorporated by reference herein in their entireties, includingthe drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR §1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing07USCNT”,created on Jul. 8, 2008, which is 172,922 bytes in size.

FIELD OF THE INVENTION

The present invention concerns methods and reagents useful in modulatinggene expression associated with Alzheimer's disease in a variety ofapplications, including use in therapeutic, diagnostic, targetvalidation, and genomic discovery applications. The present inventionconcerns compounds, compositions, and methods for the study, diagnosis,and treatment of conditions and diseases that respond to the modulationof beta-secretase (BACE), amyloid precursor protein (APP), pin-1,presenillin 1 (PS-1) and/or presenillin 2 (PS-2) gene expression and/oractivity. The present invention also concerns compounds, compositions,and methods relating to conditions and diseases that respond to themodulation of expression and/or activity of genes involved inbeta-secretase (BACE), amyloid precursor protein (APP), pin-1,presenillin 1 (PS-1) and/or presenillin 2 (PS-2) pathways. Specifically,the invention relates to small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules capable of mediating RNA interference (RNAi) againstbeta-secretase (BACE), amyloid precursor protein (APP), pin-1,presenillin 1 (PS-1) and/or presenillin 2 (PS-2) gene expression.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806; Hamiltonet al., 1999, Science, 286, 950-951). The corresponding process inplants is commonly referred to as post-transcriptional gene silencing orRNA silencing and is also referred to as quelling in fungi. The processof post-transcriptional gene silencing is thought to be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes and is commonly shared by diverse flora andphyla (Fire et al., 1999, Trends Genet., 15, 358). Such protection fromforeign gene expression may have evolved in response to the productionof double-stranded RNAs (dsRNAs) derived from viral infection or fromthe random integration of transposon elements into a host genome via acellular response that specifically destroys homologous single-strandedRNA or viral genomic RNA. The presence of dsRNA in cells triggers theRNAi response though a mechanism that has yet to be fully characterized.This mechanism appears to be different from the interferon response thatresults from dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Hamilton et al., supra; Berstein et al.,2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Hamilton et al., supra; Elbashiret al., 2001, Genes Dev., 15, 188). Dicer has also been implicated inthe excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) fromprecursor RNA of conserved structure that are implicated intranslational control (Hutvagner et al., 2001, Science, 293, 834). TheRNAi response also features an endonuclease complex, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence complementary to the antisensestrand of the siRNA duplex. Cleavage of the target RNA takes place inthe middle of the region complementary to the antisense strand of thesiRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced byintroduction of duplexes of synthetic 21-nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells. Recentwork in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J.,20, 6877) has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21-nucleotidesiRNA duplexes are most active when containing 3′-terminal dinucleotideoverhangs. Furthermore, complete substitution of one or both siRNAstrands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAiactivity, whereas substitution of the 3′-terminal siRNA overhangnucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated.Single mismatch sequences in the center of the siRNA duplex were alsoshown to abolish RNAi activity. In addition, these studies also indicatethat the position of the cleavage site in the target RNA is defined bythe 5′-end of the siRNA guide sequence rather than the 3′-end of theguide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studieshave indicated that a 5′-phosphate on the target-complementary strand ofan siRNA duplex is required for siRNA activity and that ATP is utilizedto maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well-tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition,Elbashir et al., supra, also report that substitution of siRNA with2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al.,International PCT Publication No. WO 00/44914, and Beach et al.,International PCT Publication No. WO 01/68836 preliminarily suggest thatsiRNA may include modifications to either the phosphate-sugar backboneor the nucleoside to include at least one of a nitrogen or sulfurheteroatom, however, neither application postulates to what extent suchmodifications would be tolerated in siRNA molecules, nor provides anyfurther guidance or examples of such modified siRNA. Kreutzer et al.,Canadian Patent Application No. 2,359,180, also describe certainchemical modifications for use in dsRNA constructs in order tocounteract activation of double-stranded RNA-dependent protein kinasePKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotidescontaining a 2′-O or 4′-C methylene bridge. However, Kreutzer et al.similarly fails to provide examples or guidance as to what extent thesemodifications would be tolerated in siRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain dsRNA molecules into cells for use in inhibiting geneexpression. Plaetinck et al., International PCT Publication No. WO00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific dsRNA molecules. Mello et al., International PCT PublicationNo. WO 01/29058, describe the identification of specific genes involvedin dsRNA-mediated RNAi. Deschamps Depaillette et al., International PCTPublication No. WO 99/07409, describe specific compositions consistingof particular dsRNA molecules combined with certain anti-viral agents.Waterhouse et al., International PCT Publication No. 99/53050, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA constructs foruse in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1977-1087, describespecific chemically modified siRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain dsRNAs. Echeverri et al., International PCTPublication No. WO 02/38805, describe certain C. elegans genesidentified via RNAi. Kreutzer et al., International PCT PublicationsNos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certainmethods for inhibiting gene expression using RNAi. Graham et al.,International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU4037501 describe certain vector expressed siRNA molecules. Fire et al.,U.S. Pat. No. 6,506,559, describe certain methods for inhibiting geneexpression in vitro using certain long dsRNA (greater than 25nucleotide) constructs that mediate RNAi.

McSwiggen et al., International PCT Publication No. WO 01/16312,describes nucleic acid mediated inhibition of BACE, PS-1, and PS-2expression.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating the expression of genes associated with the maintenanceor development of Alzheimer's disease and dementia, for example, BACE,APP, PIN-1, PS-1 and/or PS-2, by RNA interference (RNAi) using smallnucleic acid molecules, such as short interfering nucleic acid (siNA),short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA(miRNA), and short hairpin RNA (shRNA) molecules. In particular, theinvention relates to compounds, compositions, and methods useful formodulating the expression and activity of BACE, APP, PIN-1, PS-1 and/orPS-2 genes, or genes involved in BACE, APP, PIN-1, PS-1 and/or PS-2pathways of gene expression and/or BACE APP, PIN-1, PS-1 and/or PS-2activity by RNA interference (RNAi). Specifically, the instant inventionfeatures small nucleic acid molecules useful in RNA interference (RNAi),such as short interfering nucleic acid (siNA), short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and shorthairpin RNA (shRNA) molecules and methods used to modulate theexpression of BACE APP, PIN-1, PS-1 and/or PS-2 genes or other genesassociated with the maintenance or development of Alzheimer's diseaseand/or dementia. An siNA of the invention can be unmodified orchemically modified. An siNA of the instant invention can be chemicallysynthesized, expressed from a vector or enzymatically synthesized. Theinstant invention also features various chemically modified syntheticshort interfering nucleic acid (siNA) molecules capable of modulatingBACE APP, PIN-1, PS-1 and/or PS-2 gene expression or activity in cellsby RNA interference (RNAi). The use of chemically modified siNA improvesvarious properties of native siNA molecules through increased resistanceto nuclease degradation in vivo and/or through improved cellular uptake.Further, contrary to earlier published studies, siNA having multiplechemical modifications retains its RNAi activity. The siNA molecules ofthe instant invention provide useful reagents and methods for a varietyof therapeutic, diagnostic, target validation, genomic discovery,genetic engineering, and pharmacogenomic applications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofgene(s) encoding proteins, such as BACE, APP, PIN-1, PS-1 and/or PS-2proteins, associated with the maintenance and/or development ofAlzheimer's disease and other neurodegenerative disorders or conditionssuch as dementia, and stroke/cardiovascular accident (CVA), such asgenes encoding sequences comprising those sequences referred to byGenBank Accession Nos. shown in Table I, referred to herein generally asBACE. The description below of the various aspects and embodiments isprovided with reference to the exemplary BACE gene and BACE protein,including components or subunits thereof and variants thereof. However,the various aspects and embodiments are also directed to other geneswhich express other BACE related proteins or other proteins associatedwith Alheimer's disease, such as APP, PIN-1, PS-1 and PS-2, includingmutant genes and splice variant genes thereof. The various aspects andembodiments are also directed to other genes that are involved in BACE,APP, PIN-1, PS-1 and PS-2 mediated pathways of signal transduction orgene expression that are involved in the progression, development, ormaintenance of disease (e.g., Alzheimer's disease). These additionalgenes can be analyzed for target sites using the methods describedherein for BACE genes. Thus, the modulation of other genes and effectsof such modulation can be performed and measured as described herein. Inother words, all of the methods described herein using BACE gene as anexemplary target can be applied to other genes associated withAlzheimer's disease and dementia.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a BACE gene, for example, wherein the BACEgene comprises BACE encoding sequence.

In one embodiment, the invention features an siNA molecule having RNAiactivity against BACE RNA, wherein the siNA molecule comprises asequence complementary to an RNA having BACE encoding sequence, such asthose sequences having GenBank Accession Nos. shown in Table I. Inanother embodiment, the invention features an siNA molecule having RNAiactivity against BACE RNA, wherein the siNA molecule comprises asequence complementary to an RNA having other BACE encoding sequence,for example, mutant BACE genes, splice variants of BACE genes, variantswith conservative substitutions, and homologous BACE ligands andreceptors. Chemical modifications as shown in Tables III and IV orotherwise described herein can be applied to any siNA construct of theinvention. Furthermore, the chemically modified constructs described inTable IV can be applied to any siNA sequence of the invention.

In another embodiment, the invention features an siNA molecule havingRNAi activity against a BACE gene, wherein the siNA molecule comprisesnucleotide sequence complementary to nucleotide sequence of a BACE gene,such as those BACE sequences having GenBank Accession Nos. shown inTable I or other BACE encoding sequence, for example, mutant BACE genes,splice variants of BACE genes, BACE variants with conservativesubstitutions, and homologous BACE ligands and receptors. In anotherembodiment, an siNA molecule of the invention includes nucleotidesequence that can interact with nucleotide sequence of a BACE gene andthereby mediate silencing of BACE gene expression, for example, whereinthe siNA mediates regulation of BACE gene expression by cellularprocesses that modulate the chromatin structure of the BACE gene andprevent transcription of the BACE gene.

In another embodiment, the invention features an siNA moleculecomprising nucleotide sequence, for example, nucleotide sequence in theantisense region of the siNA molecule that is complementary to anucleotide sequence or portion of sequence of a BACE gene. In anotherembodiment, the invention features an siNA molecule comprising a region,for example, the antisense region of the siNA construct, complementaryto a sequence comprising a BACE gene sequence or a portion thereof.

In one embodiment, the antisense region of BACE siNA constructs cancomprise a sequence complementary to sequence having any of SEQ ID NOs.1-325 or 651-654. The antisense region can also comprise sequence havingany of SEQ ID NOs. 326-650, 659-662, 667-670, 675-678, 684, 688, 701,703, 705, or 708. In another embodiment, the sense region of BACEconstructs can comprise sequence having any of SEQ ID NOs. 1-325,651-658, 663-666, 671-674, 683, 687, 700, 702, 704, 706, or 707. Thesense region can comprise a sequence of SEQ ID NO. 691 and the antisenseregion can comprise a sequence of SEQ ID NO. 692. The sense region cancomprise a sequence of SEQ ID NO. 693 and the antisense region cancomprise a sequence of SEQ ID NO. 694. The sense region can comprise asequence of SEQ ID NO. 695 and the antisense region can comprise asequence of SEQ ID NO. 696. The sense region can comprise a sequence ofSEQ ID NO. 697 and the antisense region can comprise a sequence of SEQID NO. 694. The sense region can comprise a sequence of SEQ ID NO. 698and the antisense region can comprise a sequence of SEQ ID NO. 694. Thesense region can comprise a sequence of SEQ ID NO. 697 and the antisenseregion can comprise a sequence of SEQ ID NO. 699.

In one embodiment, an siNA molecule of the invention comprises any ofSEQ ID NOs. 1-708. The sequences shown in SEQ ID NOS: 1-708 are notlimiting. An siNA molecule of the invention can comprise any contiguousBACE sequence (e.g., about 19 to about 25, or about 19, 20, 21, 22, 23,24 or 25 contiguous BACE nucleotides).

In yet another embodiment, the invention features an siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siRNA construct of the invention. Furthermore, thechemically modified constructs described in Table IV can be applied toany siNA sequence of the invention.

In one embodiment of the invention an siNA molecule comprises anantisense strand having about 19 to about 29 nucleotides, wherein theantisense strand is complementary to a RNA sequence encoding a BACEprotein, and wherein said siNA further comprises a sense strand havingabout 19 to about 29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28or 29) nucleotides, and wherein said sense strand and said antisensestrand are distinct nucleotide sequences with at least about 19complementary nucleotides.

In another embodiment of the invention an siNA molecule of the inventioncomprises an antisense region having about 19 to about 29 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides, wherein theantisense region is complementary to a RNA sequence encoding a BACEprotein, and wherein said siNA further comprises a sense region havingabout 19 to about 29 nucleotides, wherein said sense region and saidantisense region comprise a linear molecule with at least about 19complementary nucleotides.

In one embodiment of the invention an siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a BACE protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a BACE gene or a portion thereof.

In another embodiment, an siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence or a portion thereof encoding a BACE protein. The siNA moleculefurther comprises a sense region, wherein said sense region comprises anucleotide sequence of a BACE gene or a portion thereof.

In one embodiment, an siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a BACE gene. Because BACEgenes can share some degree of sequence homology with each other, siNAmolecules can be designed to target a class of BACE genes (andassociated receptor or ligand genes) or alternately specific BACE genesby selecting sequences that are either shared amongst different BACEtargets or alternatively that are unique for a specific BACE target.Therefore, in one embodiment, the siNA molecule can be designed totarget conserved regions of BACE RNA sequence having homology betweenseveral BACE genes so as to target several BACE genes (e.g., differentBACE isoforms, splice variants, mutant genes etc.) with one siNAmolecule. In another embodiment, the siNA molecule can be designed totarget a sequence that is unique to a specific BACE RNA sequence due tothe high degree of specificity that the siNA molecule requires tomediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplexes containing about 19 basepairs between oligonucleotides comprising about 19 to about 25 (e.g.,about 19, 20, 21, 22, 23, 24 or 25) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplexes withoverhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3)nucleotides, for example, about 21-nucleotide duplexes with about 19base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs.

In one embodiment, the invention features one or more chemicallymodified siNA constructs having specificity for BACE expressing nucleicacid molecules, such as RNA encoding a BACE protein. Non-limitingexamples of such chemical modifications include without limitationphosphorothioate internucleotide linkages, 2′-deoxyribonucleotides,2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides,“universal base” nucleotides, “acyclic” nucleotides, 5-C-methylnucleotides, and terminal glyceryl and/or inverted deoxy abasic residueincorporation. These chemical modifications, when used in various siNAconstructs, are shown to preserve RNAi activity in cells while at thesame time, dramatically increasing the serum stability of thesecompounds. Furthermore, contrary to the data published by Parrish etal., supra, applicant demonstrates that multiple (greater than one)phosphorothioate substitutions are well-tolerated and confer substantialincreases in serum stability for modified siNA constructs.

In one embodiment, an siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, ansiNA molecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, an siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single-stranded, the percent modification can be based uponthe total number of nucleotides present in the single-stranded siNAmolecules. Likewise, if the siNA molecule is double-stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a BACEgene. In one embodiment, the double-stranded siNA molecule comprises oneor more chemical modifications and each strand of the double-strandedsiNA is about 21 nucleotides long. In one embodiment, thedouble-stranded siNA molecule does not contain any ribonucleotides. Inanother embodiment, the double-stranded siNA molecule comprises one ormore ribonucleotides. In one embodiment, each strand of thedouble-stranded siNA molecule comprises about 19 to about 23nucleotides, wherein each strand comprises at least about 19 nucleotidesthat are complementary to the nucleotides of the other strand. In oneembodiment, one of the strands of the double-stranded siNA moleculecomprises a nucleotide sequence that is complementary to a nucleotidesequence of the BACE gene or a portion thereof, and the second strand ofthe double-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the BACE gene or aportion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a BACE gene, wherein the siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence of the BACE gene or a portion thereof, and wherein the siNAfurther comprises a sense region, wherein the sense region comprises anucleotide sequence substantially similar to the nucleotide sequence ofthe BACE gene or a portion thereof. In one embodiment, the antisenseregion and the sense region each comprise about 19 to about 23nucleotides, wherein the antisense region comprises at least about 19nucleotides that are complementary to nucleotides of the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a BACE gene, wherein the siNA molecule comprises a sense region andan antisense region and wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by the BACE gene or a portion thereof and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a BACE gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule. The sense region can be connected to the antisense regionvia a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a BACE gene, wherein the siNA molecule comprises a sense region andan antisense region and wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by the BACE gene or a portion thereof and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion, wherein the siNA molecule has one or more modified pyrimidineand/or purine nucleotides. In one embodiment, the pyrimidine nucleotidesin the sense region are 2′-O-methylpyrimidine nucleotides or2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides. In anotherembodiment, the pyrimidine nucleotides in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-O-methyl purine nucleotides. Inanother embodiment, the pyrimidine nucleotides in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides. In oneembodiment, the pyrimidine nucleotides in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the antisense region are 2′-O-methyl or 2′-deoxy purinenucleotides. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of thesense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a BACE gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule, and wherein the fragment comprising the sense regionincludes a terminal cap moiety at the 5′-end, the 3′-end, or both of the5′ and 3′ ends of the fragment comprising the sense region. In oneembodiment, the terminal cap moiety is an inverted deoxy abasic moietyor glyceryl moiety. In one embodiment, each of the two fragments of thesiNA molecule comprises about 21 nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a BACE gene, wherein the siNA molecule comprises a sense region andan antisense region and wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence or aportion thereof of RNA encoded by the BACE gene and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion, and wherein the purine nucleotides present in the antisenseregion comprise 2′-deoxy-purine nucleotides. In an alternativeembodiment, the purine nucleotides present in the antisense regioncomprise 2′-O-methyl purine nucleotides. In either of the aboveembodiments, the antisense region can comprise a phosphorothioateinternucleotide linkage at the 3′ end of the antisense region.Alternatively, in either of the above embodiments, the antisense regioncan comprise a glyceryl modification at the 3′ end of the antisenseregion. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of theantisense strand (e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a BACE gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments each comprising 21 nucleotides, wherein onefragment comprises the sense region and the second fragment comprisesthe antisense region of the siNA molecule. In one embodiment, all 21nucleotides of oligonucleotide fragment are base-paired to thecomplementary nucleotides of the other fragment. In another embodiment,about 19 nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule, wherein at least two 3′ terminal nucleotides of eachfragment of the siNA molecule are not base-paired to the nucleotides ofthe other fragment of the siNA molecule. In one embodiment, each of thetwo 3′ terminal nucleotides of each fragment of the siNA molecule is a2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine. In another embodiment,about 19 nucleotides of the antisense region are base-paired to thenucleotide sequence or a portion thereof of the RNA encoded by the BACEgene. In another embodiment, 21 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the BACE gene. In any of the above embodiments, the 5′-end ofthe fragment comprising said antisense region can optionally include aphosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa BACE RNA sequence (e.g., wherein said target RNA sequence is encodedby a BACE gene or a gene involved in the BACE pathway), wherein the siNAmolecule does not contain any ribonucleotides and wherein each strand ofthe double-stranded siNA molecule is about 21 nucleotides long. Examplesof non-ribonucleotide containing siNA constructs are combinations ofstabilization chemistries shown in Table IV in any combination ofSense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab18/8, Stab 18/11, Stab 12/13, Stab 7/13, or Stab 18/13.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention and a pharmaceutically acceptable carrieror diluent.

In one embodiment, the invention features a medicament comprising ansiNA molecule of the invention.

In one embodiment, the invention features an active ingredientcomprising an siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to down-regulateexpression of a BACE gene, wherein the siNA molecule comprises one ormore chemical modifications and each strand of the double-stranded siNAis about 21 nucleotides long.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits expressionof a BACE gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of BACE RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aBACE gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of BACE RNA that encodes aprotein or portion thereof, the other strand is a sense strand whichcomprises nucleotide sequence that is complementary to a nucleotidesequence of the antisense strand and wherein a majority of thepyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aBACE gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of BACE RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification. In oneembodiment, each strand of the siNA molecule comprises about 19 to about29 nucleotides, wherein each strand comprises at least about 19nucleotides that are complementary to the nucleotides of the otherstrand. In another embodiment, the siNA molecule is assembled from twooligonucleotide fragments, wherein one fragment comprises the nucleotidesequence of the antisense strand of the siNA molecule and a secondfragment comprises nucleotide sequence of the sense region of the siNAmolecule. In yet another embodiment, the sense strand is connected tothe antisense strand via a linker molecule, such as a polynucleotidelinker or a non-nucleotide linker. In a further embodiment, thepyrimidine nucleotides present in the sense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides. In anotherembodiment, the pyrimidine nucleotides present in the sense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and the purine nucleotidespresent in the sense region are 2′-O-methyl purine nucleotides. In stillanother embodiment, the pyrimidine nucleotides present in the antisensestrand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and any purinenucleotides present in the antisense strand are 2′-deoxy purinenucleotides. In another embodiment, the antisense strand comprises oneor more 2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more2′-O-methyl purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the antisense strand are 2′-deoxy-2′-fluoropyrimidine nucleotides and any purine nucleotides present in theantisense strand are 2′-O-methyl purine nucleotides. In a furtherembodiment, wherein the sense strand comprises a 3′-end and a 5′-end, aterminal cap moiety (e.g., an inverted deoxy abasic moiety) is presentat the 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the sensestrand. In another embodiment, the antisense strand comprises aphosphorothioate internucleotide linkage at the 3′ end of the antisensestrand. In another embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aBACE gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of BACE RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein each of the two strands of the siNA molecule comprises 21nucleotides. In one embodiment, 21 nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule. In another embodiment, about 19nucleotides of each strand of the siNA molecule are base-paired to thecomplementary nucleotides of the other strand of the siNA molecule,wherein at least two 3′ terminal nucleotides of each strand of the siNAmolecule are not base-paired to the nucleotides of the other strand ofthe siNA molecule. In another embodiment, each of the two 3′ terminalnucleotides of each fragment of the siNA molecule are2′-deoxy-pyrimidines, such as 2′-deoxy-thymidine. In another embodiment,about 19 nucleotides of the antisense strand are base-paired to thenucleotide sequence of the BACE RNA or a portion thereof. In anotherembodiment, 21 nucleotides of the antisense strand are base-paired tothe nucleotide sequence of the BACE RNA or a portion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aBACE gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of BACE RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the 5′-untranslatedregion or a portion thereof of the BACE RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aBACE gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of BACE RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the BACE RNA or aportion thereof that is present in the BACE RNA.

In a non-limiting example, the introduction of chemically modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of an siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of an siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding BACE and thesense region can comprise sequence complementary to the antisenseregion. The siNA molecule can comprise two distinct strands havingcomplementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against BACE inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising abackbone modified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring or chemicallymodified, each X and Y is independently O, S, N, alkyl, or substitutedalkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl,O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W, X, Y, and Z areoptionally not all O.

The chemically modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, an siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against BACE inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I; R9 is O,S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine,guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine,2,6-diaminopurine, or any other non-naturally occurring base that can becomplementary or non-complementary to target RNA or a non-nucleosidicbase such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole,nebularine, pyridone, pyridinone, or any other non-naturally occurringuniversal base that can be complementary or non-complementary to targetRNA.

The chemically modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula II at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisensestrand, or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotides or non-nucleotides ofFormula II at the 5′-end of the sense strand, the antisense strand, orboth strands. In anther non-limiting example, an exemplary siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically modified nucleotides or non-nucleotidesof Formula II at the 3′-end of the sense strand, the antisense strand,or both strands.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against BACE inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I; R9 is O,S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such as adenine,guanine, uracil, cytosine, thymine, 2-aminoadenosine, 5-methylcytosine,2,6-diaminopurine, or any other non-naturally occurring base that can beemployed to be complementary or non-complementary to target RNA or anon-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula III at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisensestrand, or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotide(s) or non-nucleotide(s) ofFormula III at the 5′-end of the sense strand, the antisense strand, orboth strands. In anther non-limiting example, an exemplary siNA moleculeof the invention can comprise about 1 to about 5 or more (e.g., about 1,2, 3, 4, 5, or more) chemically modified nucleotide or non-nucleotide ofFormula III at the 3′-end of the sense strand, the antisense strand, orboth strands.

In another embodiment, an siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against BACE inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises a 5′-terminalphosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo; andwherein W, X, Y and Z are not all O.

In one embodiment, the invention features an siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features an siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of an siNA molecule of the invention,for example an siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against BACE inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or morephosphorothioate internucleotide linkages. For example, in anon-limiting example, the invention features a chemically modified shortinterfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 ormore phosphorothioate internucleotide linkages in one siNA strand. Inyet another embodiment, the invention features a chemically modifiedshort interfering nucleic acid (siNA) individually having about 1, 2, 3,4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in bothsiNA strands. The phosphorothioate internucleotide linkages can bepresent in one or both oligonucleotide strands of the siNA duplex, forexample in the sense strand, the antisense strand, or both strands. ThesiNA molecules of the invention can comprise one or morephosphorothioate internucleotide linkages at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the sense strand, the antisense strand,or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) consecutive phosphorothioate internucleotide linkages atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine phosphorothioate internucleotide linkages inthe sense strand, the antisense strand, or both strands. In yet anothernon-limiting example, an exemplary siNA molecule of the invention cancomprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) purine phosphorothioate internucleotide linkages in the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features an siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features an siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5 ormore, for example about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore, specifically about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features an siNA moleculecomprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotidelinkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of one or both siNA sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both siNA sequence strands, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of apyrimidine nucleotide in one or both strands of the siNA molecule cancomprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more including every internucleotide linkage of a purinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage.

In another embodiment, a chemically modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically modified, wherein each strand is about 18 to about 27(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides inlength, wherein the duplex has about 18 to about 23 (e.g., about 18, 19,20, 21, 22, or 23) base pairs, and wherein the chemical modificationcomprises a structure having any of Formulae I-VII. For example, anexemplary chemically modified siNA molecule of the invention comprises aduplex having two strands, one or both of which can be chemicallymodified with a chemical modification having any of Formulae I-VII orany combination thereof, wherein each strand consists of about 21nucleotides, each having a 2-nucleotide 3′-terminal nucleotide overhang,and wherein the duplex has about 19 base pairs. In another embodiment,an siNA molecule of the invention comprises a single-stranded hairpinstructure, wherein the siNA is about 36 to about 70 (e.g., about 36, 40,45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 toabout 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and whereinthe siNA can include a chemical modification comprising a structurehaving any of Formulae I-VII or any combination thereof. For example, anexemplary chemically modified siNA molecule of the invention comprises alinear oligonucleotide having about 42 to about 50 (e.g., about 42, 43,44, 45, 46, 47, 48, 49, or 50) nucleotides that is chemically modifiedwith a chemical modification having any of Formulae I-VII or anycombination thereof, wherein the linear oligonucleotide forms a hairpinstructure having about 19 base pairs and a 2-nucleotide 3′-terminalnucleotide overhang. In another embodiment, a linear hairpin siNAmolecule of the invention contains a stem loop motif, wherein the loopportion of the siNA molecule is biodegradable. For example, a linearhairpin siNA molecule of the invention is designed such that degradationof the loop portion of the siNA molecule in vivo can generate adouble-stranded siNA molecule with 3′-terminal overhangs, such as3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, an siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 23(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, or 23) base pairs and a 5′-terminal phosphate group thatcan be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In another embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20) base pairs, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically modified siNA molecule of the invention comprises alinear oligonucleotide having about 25 to about 35 (e.g., about 25, 26,27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemicallymodified with one or more chemical modifications having any of FormulaeI-VII or any combination thereof, wherein the linear oligonucleotideforms an asymmetric hairpin structure having about 3 to about 18 (e.g.,about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) basepairs and a 5′-terminal phosphate group that can be chemically modifiedas described herein (for example a 5′-terminal phosphate group havingFormula IV). In another embodiment, an asymmetric hairpin siNA moleculeof the invention contains a stem loop motif, wherein the loop portion ofthe siNA molecule is biodegradable. In another embodiment, an asymmetrichairpin siNA molecule of the invention comprises a loop portioncomprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric double-stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 16 to about 25 (e.g., about 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides in length, wherein the sense region is about3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18) nucleotides in length, wherein the sense region theantisense region have at least 3 complementary nucleotides, and whereinthe siNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically modified siNA molecule of the inventioncomprises an asymmetric double-stranded structure having separatepolynucleotide strands comprising sense and antisense regions, whereinthe antisense region is about 18 to about 22 (e.g., about 18, 19, 20,21, or 22) nucleotides in length and wherein the sense region is about 3to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double-stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, an siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) basepairs, and wherein the siNA can include a chemical modification, whichcomprises a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically modified siNA molecule ofthe invention comprises a circular oligonucleotide having about 42 toabout 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotidesthat is chemically modified with a chemical modification having any ofFormulae I-VII or any combination thereof, wherein the circularoligonucleotide forms a dumbbell shaped structure having about 19 basepairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R1, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I; R9 is O, S, CH2, S═O, CHF, or CF2, and either R5, R3, R8 orR13 serves as a point of attachment to the siNA molecule of theinvention.

In another embodiment, an siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-5-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a moiety having any of Formula V, VI or VII ofthe invention is at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of an siNA molecule of the invention. For example, a moietyhaving Formula V, VI or VII can be present at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the antisense strand, the sense strand, orboth antisense and sense strands of the siNA molecule. In addition, amoiety having Formula VII can be present at the 3′-end or the 5′-end ofa hairpin siNA molecule as described herein.

In another embodiment, an siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, an siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, an siNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically modified siNA comprises a sense region, where any (e.g., oneor more or all) pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically modified siNA comprises a sense region, where any (e.g., oneor more or all) pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides), wherein any nucleotides comprising a 3′-terminalnucleotide overhang that are present in said sense region are 2′-deoxynucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically modified siNA comprises a sense region, where any (e.g., oneor more or all) pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically modified siNA comprises a sense region, where any (e.g., oneor more or all) pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides), wherein any nucleotides comprising a3′-terminal nucleotide overhang that are present in said sense regionare 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically modified siNA comprises an antisense region, where any (e.g.,one or more or all) pyrimidine nucleotides present in the antisenseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and wherein any (e.g., one or more or all)purine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methylpurine nucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically modified siNA comprises an antisense region, where any (e.g.,one or more or all) pyrimidine nucleotides present in the antisenseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and wherein any (e.g., one or more or all)purine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methylpurine nucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides), wherein any nucleotides comprising a3′-terminal nucleotide overhang that are present in said antisenseregion are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically modified siNA comprises an antisense region, where any (e.g.,one or more or all) pyrimidine nucleotides present in the antisenseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and where any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-deoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically modified siNA comprises an antisense region, where any (e.g.,one or more or all) pyrimidine nucleotides present in the antisenseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and where any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against BACE inside a cell orreconstituted in vitro system, wherein the chemically modified siNAcomprises a sense region, where one or more pyrimidine nucleotidespresent in the sense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and where one or more purine nucleotides present in the sense region are2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are2′-deoxy purine nucleotides or alternately a plurality of purinenucleotides are 2′-deoxy purine nucleotides), and an antisense region,where one or more pyrimidine nucleotides present in the antisense regionare 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and where one or more purine nucleotidespresent in the antisense region are 2′-O-methyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-O-methyl purine nucleotidesor alternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). The sense region and/or the antisense region can have aterminal cap modification, such as any modification described herein orshown in FIG. 10 (i.e., an inverted deoxy abasic modification), that isoptionally present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the sense and/or antisense sequence. The sense and/orantisense region can optionally further comprise a 3′-terminalnucleotide overhang having about 1 to about 4 (e.g., about 1, 2, 3, or4) 2′-deoxynucleotides. The overhang nucleotides can further compriseone or more (e.g., 1, 2, 3, or 4) phosphorothioate internucleotidelinkages. Non-limiting examples of these chemically modified siNAs areshown in FIGS. 4 and 5 and Table III herein. In any of these describedembodiments, the purine nucleotides present in the sense region arealternatively 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides) andone or more purine nucleotides present in the antisense region are2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are2′-O-methyl purine nucleotides or alternately a plurality of purinenucleotides are 2′-O-methyl purine nucleotides). Also, in any of theseembodiments, one or more purine nucleotides present in the sense regionare alternatively purine ribonucleotides (e.g., wherein all purinenucleotides are purine ribonucleotides or alternately a plurality ofpurine nucleotides are purine ribonucleotides) and any purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). Additionally, in any of theseembodiments, one or more purine nucleotides present in the sense regionand/or present in the antisense region are alternatively selected fromthe group consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA)nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and2′-O-methyl nucleotides (e.g., wherein all purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides or alternately aplurality of purine nucleotides are selected from the group consistingof 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a Northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double-stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against BACE inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises a conjugatecovalently attached to the chemically modified siNA molecule.Non-limiting examples of conjugates contemplated by the inventioninclude conjugates and ligands described in Vargeese et al., U.S. Ser.No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein inits entirety, including the drawings. In another embodiment, theconjugate is covalently attached to the chemically modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically modifiedsiNA molecule is a poly ethylene glycol, human serum albumin, or aligand for a cellular receptor that can mediate cellular uptake.Examples of specific conjugate molecules contemplated by the instantinvention that can be attached to chemically modified siNA molecules aredescribed in Vargeese et al., U.S. Ser. No. 10/201,394, incorporated byreference herein. The type of conjugates used and the extent ofconjugation of siNA molecules of the invention can be evaluated forimproved pharmacokinetic profiles, bioavailability, and/or stability ofsiNA constructs while at the same time maintaining the ability of thesiNA to mediate RNAi activity. As such, one skilled in the art canscreen siNA constructs that are modified with various conjugates todetermine whether the siNA conjugate complex possesses improvedproperties while maintaining the ability to mediate RNAi, for example inanimal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In anotherembodiment, the nucleotide linker can be a nucleic acid aptamer. By“aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has a sequence that comprises a sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemisty 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, an siNA molecule canbe assembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, an siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the All oligonucleotide. Applicant has surprisinglyfound that the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system, wherein the siNA molecule comprises asingle-stranded polynucleotide having complementarity to a targetnucleic acid sequence. In another embodiment, the single-stranded siNAmolecule of the invention comprises a 5′-terminal phosphate group. Inanother embodiment, the single-stranded siNA molecule of the inventioncomprises a 5′-terminal phosphate group and a 3′-terminal phosphategroup (e.g., a 2′,3′-cyclic phosphate). In another embodiment, thesingle-stranded siNA molecule of the invention comprises about 19 toabout 29 nucleotides. In yet another embodiment, the single-strandedsiNA molecule of the invention comprises one or more chemically modifiednucleotides or non-nucleotides described herein. For example, all thepositions within the siNA molecule can include chemically modifiednucleotides such as nucleotides having any of Formulae I-VII, or anycombination thereof to the extent that the ability of the siNA moleculeto support RNAi activity in a cell is maintained.

In one embodiment, an siNA molecule of the invention is asingle-stranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system, wherein the siNA molecule comprises asingle-stranded polynucleotide having complementarity to a targetnucleic acid sequence, wherein one or more pyrimidine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and wherein any purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides), and a terminal cap modification, suchas any modification described herein or shown in FIG. 10, that isoptionally present at the 3′-end and/or the 5′-end. The siNA optionallyfurther comprises about 1 to about 4 (e.g., about 1, 2, 3, or 4)terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, whereinthe terminal nucleotides can further comprise one or more (e.g., 1, 2,3, or 4) phosphorothioate internucleotide linkages, and wherein the siNAoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group. In any of these embodiments, any purinenucleotides present in the antisense region are alternatively 2′-deoxypurine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxypurine nucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides). Also, in any of these embodiments, anypurine nucleotides present in the siNA (i.e., purine nucleotides presentin the sense and/or antisense region) can alternatively be lockednucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides areLNA nucleotides or alternately a plurality of purine nucleotides are LNAnucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA are alternatively 2′-methoxyethyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-methoxyethyl purinenucleotides or alternately a plurality of purine nucleotides are2′-methoxyethyl purine nucleotides).

In another embodiment, any modified nucleotides present in thesingle-stranded siNA molecules of the invention comprise modifiednucleotides having properties or characteristics similar to naturallyoccurring ribonucleotides. For example, the invention features siNAmolecules including modified nucleotides having a Northern conformation(e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Assuch, chemically modified nucleotides present in the single-strandedsiNA molecules of the invention are preferably resistant to nucleasedegradation while at the same time maintaining the capacity to mediateRNAi.

In one embodiment, the invention features a method for modulating theexpression of a BACE gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the BACE gene; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the BACE gene inthe cell.

In one embodiment, the invention features a method for modulating theexpression of a BACE gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the BACE gene and wherein the sense strand sequence of the siNAcomprises a sequence identical or substantially similar to the sequenceof the target RNA; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the BACE gene inthe cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one BACE gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the BACE genes; and (b) introducing the siNAmolecules into a cell under conditions suitable to modulate theexpression of the BACE genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more BACE genes within a cell comprising: (a)synthesizing two or more siNA molecules of the invention, which can bechemically modified, wherein the siNA strands comprise sequencescomplementary to RNA of the BACE genes and wherein the sense strandsequences of the siNAs comprise sequences identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecules into a cell under conditions suitable to modulate theexpression of the BACE genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one BACE gene within a cell comprising: (a)synthesizing [a] siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the BACE genes and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecule[s] into a cell under conditions suitable to modulate theexpression of the BACE genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. In one embodiment, theinvention features a method of modulating the expression of a BACE genein a tissue explant comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the BACE gene; and(b) introducing the siNA molecule into a cell of the tissue explantderived from a particular organism under conditions suitable to modulatethe expression of the BACE gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of theBACE gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a BACE gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the BACE gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequence of the target RNA; and (b) introducing the siNAmolecule into a cell of the tissue explant derived from a particularorganism under conditions suitable to modulate the expression of theBACE gene in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the BACE gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one BACE gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically modified, wherein one of the siNA strands comprises asequence complementary to RNA of the BACE genes; and (b) introducing thesiNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate the expressionof the BACE genes in the tissue explant. In another embodiment, themethod further comprises introducing the tissue explant back into theorganism the tissue was derived from or into another organism underconditions suitable to modulate the expression of the BACE genes in thatorganism.

In one embodiment, the invention features a method of modulating theexpression of a BACE gene in an organism comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the BACE gene; and (b) introducing the siNA molecule into theorganism under conditions suitable to modulate the expression of theBACE gene in the organism.

In another embodiment, the invention features a method of modulating theexpression of more than one BACE gene in an organism comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the BACE genes; and (b) introducing the siNAmolecules into the organism under conditions suitable to modulate theexpression of the BACE genes in the organism.

In one embodiment, the invention features a method for modulating theexpression of a BACE gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the BACE gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate theexpression of the BACE gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one BACE gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the BACE gene; and (b) contacting the siNAmolecule with a cell in vitro or in vivo under conditions suitable tomodulate the expression of the BACE genes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a BACE gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the BACE gene; and (b) contacting the siNAmolecule with a cell of the tissue explant derived from a particularorganism under conditions suitable to modulate the expression of theBACE gene in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the BACE gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one BACE gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically modified, wherein the siNA comprises a single-strandedsequence having complementarity to RNA of the BACE gene; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate theexpression of the BACE genes in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of theBACE genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a BACE gene in an organism comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the BACE gene; and (b) introducing the siNAmolecule into the organism under conditions suitable to modulate theexpression of the BACE gene in the organism.

In another embodiment, the invention features a method of modulating theexpression of more than one BACE gene in an organism comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein the siNA comprises a single-stranded sequence havingcomplementarity to RNA of the BACE gene; and (b) introducing the siNAmolecules into the organism under conditions suitable to modulate theexpression of the BACE genes in the organism.

In one embodiment, the invention features a method of modulating theexpression of a BACE gene in an organism comprising contacting theorganism with an siNA molecule of the invention under conditionssuitable to modulate the expression of the BACE gene in the organism.

In another embodiment, the invention features a method of modulating theexpression of more than one BACE gene in an organism comprisingcontacting the organism with one or more siNA molecules of the inventionunder conditions suitable to modulate the expression of the BACE genesin the organism.

The siNA molecules of the invention can be designed to down-regulate orinhibit target (BACE) gene expression through RNAi targeting of avariety of RNA molecules. In one embodiment, the siNA molecules of theinvention are used to target various RNAs corresponding to a targetgene. Non-limiting examples of such RNAs include messenger RNA (mRNA),alternate RNA splice variants of target gene(s), post-transcriptionallymodified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNAtemplates. If alternate splicing produces a family of transcripts thatare distinguished by usage of appropriate exons, the instant inventioncan be used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith siNA molecules of the invention. Such applications can beimplemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as BACE family genes. As such, siNA molecules targetingmultiple BACE targets can provide increased therapeutic effect. Inaddition, siNA can be used to characterize pathways of gene function ina variety of applications. For example, the present invention can beused to inhibit the activity of target gene(s) in a pathway to determinethe function of uncharacterized gene(s) in gene function analysis, mRNAfunction analysis, or translational analysis. The invention can be usedto determine potential target gene pathways involved in various diseasesand conditions toward pharmaceutical development. The invention can beused to understand pathways of gene expression involved in, for example,the progression and/or maintenance of Alzheimer's disease.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down-regulate the expression of gene(s) that encode RNA referredto by Genbank Accession numbers., for example BACE genes encoding RNAsequence(s) referred to herein by Genbank Accession number, for example,Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 19 to about 25 (e.g., about 19, 20, 21,22, 23, 24, or 25) nucleotides in length. In one embodiment, the assaycan comprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. In another embodiment, fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, Northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4N, where N represents the numberof base paired nucleotides in each of the siNA construct strands (e.g.,for an siNA construct having 21 nucleotide sense and antisense strandswith 19 base pairs, the complexity would be 4¹⁹); and (b) assaying thesiNA constructs of (a) above, under conditions suitable to determineRNAi target sites within the target BACE RNA sequence. In anotherembodiment, the siNA molecules of (a) have strands of a fixed length,for example about 23 nucleotides in length. In yet another embodiment,the siNA molecules of (a) are of differing length, for example havingstrands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or25) nucleotides in length. In one embodiment, the assay can comprise areconstituted in vitro siNA assay as described in Example 7 herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. In another embodiment, fragments of BACERNA are analyzed for detectable levels of cleavage, for example by gelelectrophoresis, Northern blot analysis, or RNAse protection assays, todetermine the most suitable target site(s) within the target BACE RNAsequence. The target BACE RNA sequence can be obtained as is known inthe art, for example, by cloning and/or transcription for in vitrosystems, and by cellular expression in in vivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 19 to about25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. Inone embodiment, the assay can comprise a reconstituted in vitro siNAassay as described herein. In another embodiment, the assay can comprisea cell culture system in which target RNA is expressed. Fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, Northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by an siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention, which can be chemically modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for reducing orpreventing tissue rejection in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thereduction or prevention of tissue rejection in the subject.

In another embodiment, the invention features a method for validating aBACE gene target, comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of a BACE target gene;(b) introducing the siNA molecule into a cell, tissue, or organism underconditions suitable for modulating expression of the BACE target gene inthe cell, tissue, or organism; and (c) determining the function of thegene by assaying for any phenotypic change in the cell, tissue, ororganism.

In another embodiment, the invention features a method for validating aBACE target comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of a BACE target gene;(b) introducing the siNA molecule into a biological system underconditions suitable for modulating expression of the BACE target gene inthe biological system; and (c) determining the function of the gene byassaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human,animal, plant, insect, bacterial, viral or other sources, wherein thesystem comprises the components required for RNAi activity. The term“biological system” includes, for example, a cell, tissue, or organism,or extract thereof. The term biological system also includesreconstituted RNAi systems that can be used in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing an siNAmolecule of the invention, which can be chemically modified, that can beused to modulate the expression of a BACE target gene in a biologicalsystem, including, for example, in a cell, tissue, or organism. Inanother embodiment, the invention features a kit containing more thanone siNA molecule of the invention, which can be chemically modified,that can be used to modulate the expression of more than one BACE targetgene in a biological system, including, for example, in a cell, tissue,or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically modified. Inanother embodiment, the cell containing an siNA molecule of theinvention is a mammalian cell. In yet another embodiment, the cellcontaining an siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of an siNA molecule of the invention,which can be chemically modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing ansiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for exampleunder hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizingan siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against a BACE, wherein the siNA construct comprises one or morechemical modifications, for example, one or more chemical modificationshaving any of Formulae I-VII or any combination thereof that increasesthe nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formulae I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In one embodiment, the invention features siNA constructs that mediateRNAi against a BACE, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formulae I-VII or any combination thereof intoan siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against a BACE, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against a BACE, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formulae I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formulae I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against a BACE, wherein the siNA construct comprises one or morechemical modifications described herein that modulate the polymeraseactivity of a cellular polymerase capable of generating additionalendogenous siNA molecules having sequence homology to the chemicallymodified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaeI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically modified siNAmolecule.

In one embodiment, the invention features chemically modified siNAconstructs that mediate RNAi against a BACE in a cell, wherein thechemical modifications do not significantly effect the interaction ofsiNA with a target RNA molecule, DNA molecule and/or proteins or otherfactors that are essential for RNAi in a manner that would decrease theefficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against BACE comprising (a)introducing nucleotides having any of Formulae I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingimproved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against a BACEtarget RNA comprising (a) introducing nucleotides having any of FormulaeI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against a BACEtarget DNA comprising (a) introducing nucleotides having any of FormulaeI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against a BACE, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the cellularuptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against BACE with improved cellular uptake comprising (a)introducing nucleotides having any of Formulae I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingimproved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against a BACE, wherein the siNA construct comprises one or morechemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability, comprising (a)introducing a conjugate into the structure of an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include an siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Bass,2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498;and Kreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus etal., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).Non-limiting examples of siNA molecules of the invention are shown inFIGS. 4-6, and Tables II and III herein. For example the siNA can be adouble-stranded polynucleotide molecule comprising self-complementarysense and antisense regions, wherein the antisense region comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be assembled from twoseparate oligonucleotides, where one strand is the sense strand and theother is the antisense strand, wherein the antisense and sense strandsare self-complementary (i.e. each strand comprises nucleotide sequencethat is complementary to nucleotide sequence in the other strand; suchas where the antisense strand and sense strand form a duplex ordouble-stranded structure, for example wherein the double-strandedregion is about 19 base pairs); the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. Alternatively, the siNA is assembled froma single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s). The siNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single-stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single-stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al.,2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interations, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide, chemicallymodified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), andothers. In addition, as used herein, the term RNAi is meant to beequivalent to other terms used to describe sequence specific RNAinterference, such as post transcriptional gene silencing, translationalinhibition, or epigenetics. For example, siNA molecules of the inventioncan be used to epigenetically silence genes at both thepost-transcriptional level and the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure to alter gene expression (see, for example,Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complimentary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 19 to about 22 nucleotides) and a loop region comprisingabout 4 to about 8 nucleotides, and a sense region having about 3 toabout 18 nucleotides that are complementary to the antisense region. Theasymmetric hairpin siNA molecule can also comprise a 5′-terminalphosphate group that can be chemically modified. The loop portion of theasymmetric hairpin siNA molecule can comprise nucleotides,non-nucleotides, linker molecules, or conjugate molecules as describedherein.

By “asymmetric duplex” as used herein is meant an siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complimentarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 19 to about 22 nucleotides) and asense region having about 3 to about 18 nucleotides that arecomplementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. By “gene” or“target gene” is meant, a nucleic acid that encodes an RNA, for example,nucleic acid sequences including, but not limited to, structural genesencoding a polypeptide. The target gene can be a gene derived from acell, an endogenous gene, a transgene, or exogenous genes such as genesof a pathogen, for example a virus, which is present in the cell afterinfection thereof. The cell containing the target gene can be derivedfrom or contained in any organism, for example a plant, animal,protozoan, virus, bacterium, or fungus. Non-limiting examples of plantsinclude monocots, dicots, or gymnosperms. Non-limiting examples ofanimals include vertebrates or invertebrates. Non-limiting examples offungi include molds or yeasts.

By “BACE” or “beta secretase” as used herein is meant, any protein,peptide, or polypeptide, having beta-secretase activity, such as thatinvolved in generating beta-amyloid. The term BACE also refers tonucleotide sequences that encode BACE protein or a portion, component orsubunit thereof. The term BACE is also meant to include mutant BACEgene/protein sequences and variant BACE gene/protein sequences, as wellas other sequences described herein.

By “APP” or “amyloid precurson protein” as used herein is meant, anyprotein, peptide, or polypeptide that is processed to generatebeta-amyloid. The term APP also refers to nucleotide sequences thatencode amyloid precurson protein.

By “presenillin” or “PS”, e.g., “PS-1” or “PS-2” as used herein ismeant, any protein, peptide, or polypeptide having gamma-secretaseactivity, such as that involved in generating beta-amyloid. The termpresenillin also refers to nucleotide sequences that encode presenillinprotein, e.g., PS-1 or PS-2.

By “PIN-1” as used herein is meant, any protein, peptide, or polypeptidehaving peptidyl-prolyl cis/trans isomerase activity, such as thatinvolved in the development of Neurofibrillary Tangles. The term PIN-1also refers to nucleotide sequences that encode PIN-1 protein.

By “highly conserved sequence region” is meant, a nucleotide sequence ofone or more regions in a target gene does not vary significantly fromone generation to the other or from one biological system to the other.

By “sense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of an siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of an siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%,90%, and 100% complementary). “Perfectly complementary” means that allthe contiguous residues of a nucleic acid sequence will hydrogen bondwith the same number of contiguous residues in a second nucleic acidsequence.

The siRNA molecules of the invention represent a novel therapeuticapproach to treat a variety of pathologic neurodegenerative indicationsand conditions, including Alzheimer's disease, dementia, stroke (CVA),and any other diseases or conditions that are related to the levels ofBACE in a cell or tissue, alone or in combination with other therapies.The reduction of BACE expression (specifically BACE RNA levels) and thusreduction in the level of the respective protein relieves, to someextent, the symptoms of the disease or condition.

In one embodiment of the present invention, each sequence of an siNAmolecule of the invention is independently about 18 to about 24nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22,23, or 24 nucleotides in length. In another embodiment, the siNAduplexes of the invention independently comprise about 17 to about 23base pairs (e.g., about 17, 18, 19, 20, 21, 22 or 23). In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., 38, 39, 40,41, 42, 43 or 44) nucleotides in length and comprising about 16 to about22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNAmolecules of the invention are shown in Tables II and III and FIGS. 4and 5. Exemplary synthetic siNA molecules of the invention are shown inTable III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through injection, infusion pump or stent, with or without theirincorporation in biopolymers. In particular embodiments, the nucleicacid molecules of the invention comprise sequences shown in TablesII-III and/or FIGS. 4-5. Examples of such nucleic acid molecules consistessentially of sequences defined in these tables and figures.Furthermore, the chemically modified constructs described in Table IVcan be applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to treatdiseases or conditions discussed herein (e.g., Alzheimer's disease andother neurodegenerative conditions). For example, to treat a particulardisease or condition, the siNA molecules can be administered to asubject or can be administered to other appropriate cells evident tothose skilled in the art, individually or in combination with one ormore drugs under conditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to treat conditions or diseases discussedabove. For example, the described molecules could be used in combinationwith one or more known therapeutic agents to treat a disease orcondition. Non-limiting examples of other therapeutic agents that can bereadily combined with an siNA molecule of the invention are enzymaticnucleic acid molecules, allosteric nucleic acid molecules, antisense,decoy, or aptamer nucleic acid molecules, antibodies such as monoclonalantibodies, small molecules, and other organic and/or inorganiccompounds including metals, salts and ions.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of an siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms an siNA molecule. Non-limitingexamples of such expression vectors are described in Paul et al., 2002,Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, NatureBiotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500;and Novina et al., 2002, Nature Medicine, advance online publicationdoi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for an siNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for exampleGenbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form ansiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety and wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”connects the (N N) nucleotides in the antisense strand. The antisensestrand of constructs A-F comprise sequence complementary to any targetnucleic acid sequence of the invention. Furthermore, when a glycerylmoiety (L) is present at the 3′-end of the antisense strand for anyconstruct shown in FIG. 4 A-F, the modified internucleotide linkage isoptional.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s” connects the (N N) nucleotidesin the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s” connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s” connects the (N N) nucleotides in theantisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s” connects the (N N) nucleotides in the antisense strand.

FIG. 5A-F shows non-limiting examples of specific chemically modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a BACE siNA sequence. Such chemicalmodifications can be applied to any sequence herein, such as any BACEsequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have about 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined BACE target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, which is followed by a loop sequence of defined sequence (X),comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in an siNA transcript having specificity for a BACE targetsequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double-stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined BACE target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, and which is followed by a 3′-restriction site (R2) which isadjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double-stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows a non-limiting example of reduction of BACE mRNA in A549cells mediated by siNAs that target BACE mRNA. A549 cells weretransfected with 0.25 ug/well of lipid complexed with 25 nM siNA. Ascreen of siNA constructs comprising ribonucleotides and 3′-terminaldithymidine caps was compared to untreated cells, scrambled siNA controlconstructs (Scram1 and Scram2), and cells transfected with lipid alone(transfection control). As shown in the figure, all of the siNAconstructs show significant reduction of BACE RNA expression.

FIG. 13 shows a non-limiting example of reduction of BACE mRNA in A549cells mediated by siNAs that target BACE mRNA using chemically modifiedsiNA constructs. A549 cells were transfected with 0.25 ug/well of lipidcomplexed with 25 nM siNA. A lead siNA construct (31007/31083) chosenfrom the screen described in FIG. 12 was further modified using chemicalmodifications described in Table IV herein. Chemically modifiedconstructs having Stab 4/5 chemistry (31378/31381) and Stab 7/11chemistry (31384/31387) (see Table IV) were tested for efficacy comparedto matched chemistry inverted controls (sequences are shown in TableIII). The original lead siNA construct (31007/31083) and the Stab 4/5and Stab 7/11 constructs were compared to untreated cells, scrambledsiNA control constructs (Scram1 and Scram2), and cells transfected withlipid alone (transfection control). As shown in the figure, the originallead construct and the Stab 4/5 and Stab 7/11 modified siNA constructsall show significant reduction of BACE RNA expression.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of NucleicAcid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or an siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates (Elbashir et al., 2001, EMBO J., 20, 6877) has revealed certainrequirements for siRNA length, structure, chemical composition, andsequence that are essential to mediate efficient RNAi activity. Thesestudies have shown that 21-nucleotide siRNA duplexes are most activewhen containing two 2-nucleotide 3′-terminal dinucleotide overhangs.Furthermore, substitution of one or both siRNA strands with 2′-deoxy(2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity, whereassubstitution of the 3′-terminal siRNA nucleotides with 2′-deoxynucleotides (2′-H) was shown to be tolerated. Mismatch sequences in thecenter of the siRNA duplex were also shown to abolish RNAi activity. Inaddition, these studies also indicate that the position of the cleavagesite in the target RNA is defined by the 5′-end of the siRNA guidesequence rather than the 3′-end of the guide sequence (Elbashir et al.,2001, EMBO J., 20, 6877). Other studies have indicated that a5′-phosphate on the target-complementary strand of an siRNA duplex isrequired for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309)however, siRNA molecules lacking a 5′-phosphate are active whenintroduced exogenously, suggesting that 5′-phosphorylation of siRNAconstructs may occur in vivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™. Burdick &Jackson Synthesis Grade acetonitrile is used directly from the reagentbottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made upfrom the solid obtained from American International Chemical, Inc.Alternately, for the introduction of phosphorothioate linkages, Beaucagereagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile)is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & JacksonSynthesis Grade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10minutes. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 hour, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO:1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperature.TEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenpre-washed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

An siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment comprises the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211,3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to an siNA molecule of the invention or thesense and antisense strands of an siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes; nucleic acid molecules coupled with known smallmolecule modulators; or intermittent treatment with combinations ofmolecules, including different motifs and/or other chemical orbiological molecules). The treatment of subjects with siNA molecules canalso include combinations of different types of nucleic acid molecules,such as enzymatic nucleic acid molecules (ribozymes), allozymes,antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect an siNA molecule of the invention comprises one ormore 5′ and/or a 3′-cap structure, for example on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap include, but are notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and suitable heterocyclic groups include furanyl, thienyl, pyridyl,pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl andthe like, all optionally substituted. An “amide” refers to an—C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An“ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylarylor hydrogen.

“Nucleotide” as used herein, and as recognized in the art, includesnatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1 position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1 carbon of β-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-V11 and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′—NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

An siNA molecule of the invention can be adapted for use to treat avariety of neurodegenerative diseases, including Alzheimer's disease,dementia, stroke (CVA), and any other diseases or conditions that arerelated to the levels of BACE in a cell or tissue, alone or incombination with other therapies. For example, an siNA molecule cancomprise a delivery vehicle, including liposomes, for administration toa subject, carriers and diluents and their salts, and/or can be presentin pharmaceutically acceptable formulations. Methods for the delivery ofnucleic acid molecules are described in Akhtar et al., 1992, Trends CellBio., 2, 139; Delivery Strategies for Antisense OligonucleotideTherapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol.,16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137,165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all ofwhich are incorporated herein by reference. Beigelman et al., U.S. Pat.No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe thegeneral methods for delivery of nucleic acid molecules. These protocolscan be utilized for the delivery of virtually any nucleic acid molecule.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTPublication No. WO 03/47518; and Wang, International PCT Publication No.WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres(see for example U.S. Pat. No. 6,447,796 and US Patent ApplicationPublication No. US 2002130430), biodegradable nanocapsules, andbioadhesive microspheres, or by proteinaceous vectors (O'Hare andNormand, International PCT Publication No. WO 00/53722). Alternatively,the nucleic acid/vehicle combination is locally delivered by directinjection or by use of an infusion pump. Direct injection of the nucleicacid molecules of the invention, whether subcutaneous, intramuscular, orintradermal, can take place using standard needle and syringemethodologies, or by needle-free technologies such as those described inConry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry et al.,International PCT Publication No. WO 99/31262. The molecules of theinstant invention can be used as pharmaceutical agents. Pharmaceuticalagents prevent, modulate the occurrence, or treat (alleviate a symptomto some extent, preferably all of the symptoms) of a disease state in asubject.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedinto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, or by injection. Such forms should notprevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes that lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes exposes the siNA molecules of theinvention to an accessible diseased tissue. The rate of entry of a druginto the circulation has been shown to be a function of molecular weightor size. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach can provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cells producing excess BACE.

By “pharmaceutically acceptable formulation” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge,Mass.); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate, which can deliver drugs across the blood brainbarrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount from about 0.1 mg/kg to about 100mg/kg body weight/day of active ingredients is administered dependentupon potency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain about 1 mg to about 500 mg of an activeingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention provides compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatennary or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; propulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe etal., 1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, Gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pats.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from a subject followed by reintroduction into thesubject, or by any other means that would allow for introduction intothe desired target cell (for a review see Couture et al., 1996, TIG.,12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of ansiNA duplex, or a single self-complementary strand that self hybridizesinto an siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi: 10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention,and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. U S A, 87, 6743-7;Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al.,1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol.,10, 4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S.A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of an siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of an siNA molecule, wherein the sequenceis operably linked to the 3′-end of the open reading frame and whereinthe sequence is operably linked to the initiation region, the intron,the open reading frame and the termination region in a manner whichallows expression and/or delivery of the siNA molecule.

BACE Biology and Biochemistry

Alzheimer's disease is characterized by the progressive formation ofinsoluble plaques and vascular deposits in the brain consisting of the 4kD amyloid β peptide (Aβ). These plaques are characterized by dystrophicneurites that show profound synaptic loss, neurofibrillary tangleformation, and gliosis. Aβ arises from the proteolytic cleavage of thelarge type I transmembrane protein, β-amyloid precursor protein (APP)(Kang et al., 1987, Nature, 325, 733). Processing of APP to generate Aβrequires two sites of cleavage by a β-secretase and a γ-secretase.β-secretase cleavage of APP results in the cytoplasmic release of a 100kD soluble amino-terminal fragment, APPsβ, leaving behind a 12 kDtransmembrane carboxy-terminal fragment, C99. Alternately, APP can becleaved by a α-secretase to generate cytoplasmic APPsα0 andtransmembrane C83 fragments. Both remaining transmembrane fragments, C99and C83, can be further cleaved by a 7-secretase, leading to the releaseand secretion of Alzheimer's related Aβ and a non-pathogenic peptide,p3, respectively (Vassar et al., 1999, Science, 286, 735-741). Earlyonset familial Alzheimer's disease is characterized by mutant APPprotein with a Met to Leu substitution at position P1, characterized asthe “Swedish” familial mutation (Mullan et al., 1992, Nature Genet., 1,345). This APP mutation is characterized by a dramatic enhancement inβ-secretase cleavage (Citron et al., 1992, Nature, 360, 672).

The identification of β-secretase and γ-secretase constituents involvedin the release of β-amyloid protein is of primary importance in thedevelopment of treatment strategies for Alzheimer's disease.Characterization of α-secretase is also important in this regard sinceα-secretase cleavage may compete with β-secretase cleavage resulting inchanges in the relative amounts of non-pathogenic and pathogenic proteinproduction. Involvement of the two metalloproteases, ADAM 10 and TACE,has been demonstrated in α-cleavage of AAP (Buxbaum et al., 1999, J.Biol. Chem., 273, 27765, and Lammich et al., 1999, Proc. Natl. Acad.Sci. U.S.A., 96, 3922). Studies of 7-secretase activity havedemonstrated presenilin dependence (De Stooper et al., 1998, Nature,391, 387, and De Stooper et al., 1999, Nature, 398, 518), and as such,presenilins have been proposed as 7-secretase even though presenilindoes not present proteolytic activity (Wolfe et al., 1999, Nature, 398,513).

Studies have shown β-secretase cleavage of AAP by the transmembraneaspartic protease beta site APP cleaving enzyme, BACE (Vassar et al.,supra). While other potential candidates for β-secretase have beenproposed (for review see Evin et al., 1999, Proc. Natl. Acad. Sci.U.S.A., 96, 3922), none have demonstrated the full range ofcharacteristics expected from this enzyme. Studies have shown that BACEexpression and localization are as expected for β-secretase, that BACEoverexpression in cells results in increased β-secretase cleavage of APPand Swedish APP, that isolated BACE demonstrates site specificproteolytic activity on APP derived peptide substrates, and thatantisense mediated endogenous BACE inhibition results in dramaticallyreduced β-secretase activity (Vassar et al., supra).

Current treatment strategies for Alzheimer's disease rely on either theprevention or the alleviation of symptoms and/or the slowing down ofdisease progression. Two drugs approved in the treatment of Alzheimer's,donepezil (Aricept®) and tacrine (Cognex®), both cholinomimetics,attempt to slow the loss of cognitive ability by increasing the amountof acetylcholine available to the brain. Antioxidant therapy through theuse of antioxidant compounds such as alpha-tocopherol (vitamin E),melatonin, and selegeline (Eldepryl®) attempt to slow diseaseprogression by minimizing free radical damage. Estrogen replacementtherapy is thought to incur a possible preventative benefit in thedevelopment of Alzheimer's disease based on limited data. The use ofanti-inflammatory drugs may be associated with a reduced risk ofAlzheimer's as well. Calcium channel blockers such as Nimodipine® areconsidered to have a potential benefit in treating Alzheimer's diseasedue to protection of nerve cells from calcium overload, therebyprolonging nerve cell survival. Nootropic compounds, such asacetyl-L-carnitine (Alcar®) and insulin, have been proposed to have somebenefit in treating Alzheimer's due to enhancement of cognitive andmemory function based on cellular metabolism.

Whereby the above treatment strategies can all improve quality of lifein Alzheimer's patients, there exists an unmet need in the comprehensivetreatment and prevention of this disease. As such, there exists the needfor therapeutics effective in reversing the physiological changesassociated with Alzheimer's disease, specifically, therapeutics that caneliminate and/or reverse the deposition of amyloid β peptide. The use ofcompounds, such as small nucleic acid molecules (e.g., short interferingnucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA(dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) moleculescapable of mediating RNA interference (RNAi)), to modulate theexpression of proteases that are instrumental in the release of amyloidβ peptide, namely β-secretase (BACE), γ-secretase (presenilin), and theamyloid precursor protein (APP), is of therapeutic significance.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of an siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H₂O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H₂O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H₂O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H₂O followed by 1 CV 1M NaCl and additional H₂O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Sites in an RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Tables II and III). Ifterminal TT residues are desired for the sequence (as described inparagraph 7), then the two 3′ terminal nucleotides of both the sense andantisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.

In an alternate approach, a pool of siNA constructs specific to a BACEtarget sequence is used to screen for target sites in cells expressingBACE RNA, such A549 cells, 7PA2 Chinese hamster ovary (CHO) cells orAPPsw (Swedish type amyloid precursor protein expressing) cells. Thegeneral strategy used in this approach is shown in FIG. 9. Anon-limiting example of such as pool is a pool comprising sequenceshaving sense sequences comprising SEQ ID NOs. 1-325, 651-658, 663-666,671-674, 683 and 687, and antisense sequences comprising SEQ ID NOs.326-650, 659-662, 667-670, 675-678, 684, and 688, respectively. Cellsexpressing BACE (e.g., A549 cells) are transfected with the pool of siNAconstructs, and cells that demonstrate a phenotype associated with BACEinhibition are sorted. The pool of siNA constructs can be expressed fromtranscription cassettes inserted into appropriate vectors (see forexample FIG. 7 and FIG. 8). The siNA from cells demonstrating a positivephenotypic change (e.g., decreased proliferation, decreased BACE mRNAlevels or decreased BACE protein expression), are sequenced to determinethe most suitable target site(s) within the target BACE RNA sequence.

Example 4 BACE Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the BACE RNAtarget and optionally prioritizing the target sites on the basis offolding (structure of any given sequence analyzed to determine siNAaccessibility to the target), by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O—Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example, by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Scaringe supra, Usman et al., U.S. Pat. No.5,831,071, U.S. Pat. No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellonet al., U.S. Pat. No. 6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No.6,303,773, all of which are incorporated by reference herein in theirentireties.

Additionally, deprotection conditions can be modified to provide thebest possible yield and purity of siNA constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi in Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting BACE RNA targets. The assaycomprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with BACE target RNA. A Drosophila extract derived fromsyncytial blastoderm is used to reconstitute RNAi activity in vitro.Target RNA is generated via in vitro transcription from an appropriateBACE expressing plasmid using T7 RNA polymerase or via chemicalsynthesis as described herein. Sense and antisense siNA strands (forexample 20 uM each) are annealed by incubation in buffer (such as 100 mMpotassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for1 min. at 90° C. followed by 1 hour at 37° C., then diluted in lysisbuffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate). Annealing can be monitored by gelelectrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophila lysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10%[vol/vol] lysis buffer containing siNA (10 nM final concentration). Thereaction mixture also contains 10 mM creatine phosphate, 10 ug.mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25× Passive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG 50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by Phosphor Imager® quantitation ofbands representing intact control RNA or RNA from control reactionswithout siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites the BACERNA target for siNA mediated RNAi cleavage, wherein a plurality of siNAconstructs are screened for RNAi mediated cleavage of the BACE RNAtarget, for example, by analyzing the assay reaction by electrophoresisof labeled target RNA, or by Northern blotting, as well as by othermethodology well known in the art.

Example 7 Nucleic Acid Inhibition of BACE Target RNA in Vivo

siNA molecules targeted to the human BACE RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within theBACE RNA are given in Tables II and III.

Two formats are used to test the efficacy of siNAs targeting BACE.First, the reagents are tested in cell culture using, for example, A549cells, 7PA2 Chinese hamster ovary (CHO) cells or APPsw (Swedish typeamyloid precursor protein expressing) cells to determine the extent ofRNA and protein inhibition. siNA reagents (e.g., see Tables II and III)are selected against the BACE target as described herein. RNA inhibitionis measured after delivery of these reagents by a suitable transfectionagent to, for example, A549 cells, 7PA2 Chinese hamster ovary (CHO)cells or APPsw (Swedish type amyloid precursor protein expressing)cells. Relative amounts of target RNA are measured versus actin usingreal-time PCR monitoring of amplification (e.g., ABI 7700 Taqman®). Acomparison is made to a mixture of oligonucleotide sequences made tounrelated targets or to a randomized siNA control with the same overalllength and chemistry, but randomly substituted at each position. Primaryand secondary lead reagents are chosen for the target and optimizationperformed. After an optimal transfection agent concentration is chosen,a RNA time-course of inhibition is performed with the lead siNAmolecule. In addition, a cell-plating format can be used to determineRNA inhibition.

Delivery of siNA to Cells

Cells (e.g., A549 cells, 7PA2, CHO, or APPsw cells) are seeded, forexample, at 1×10⁵ cells per well of a six-well dish in EGM-2(BioWhittaker) the day before transfection. siNA (final concentration,for example 20 nM) and cationic lipid (e.g., final concentration 2μg/ml) are complexed in EGM basal media (Biowhittaker) at 37° C. for 30mins in polystyrene tubes. Following vortexing, the complexed siNA isadded to each well and incubated for the times indicated. For initialoptimization experiments, cells are seeded, for example, at 1×10³ in 96well plates and siNA complex added as described. Efficiency of deliveryof siNA to cells is determined using a fluorescent siNA complexed withlipid. Cells in 6-well dishes are incubated with siNA for 24 hours,rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at roomtemperature. Uptake of siNA is visualized using a fluorescentmicroscope.

Taqman and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For Taqman analysis, dual-labeled probes aresynthesized with the reporter dye, FAM or JOE, covalently linked at the5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-stepRT-PCR amplifications are performed on, for example, an ABI PRISM 7700Sequence Detector using 50 μl reactions consisting of 10 μl total RNA,100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1× TaqManPCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM eachdATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25UAmpliTaq Gold (PE-Applied Biosystems) and 10U M-MLV ReverseTranscriptase (Promega). The thermal cycling conditions can consist of30 min at 48° C., 10 min at 95° C., followed by 40 cycles of 15 sec at95° C. and 1 min at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/rxn) and normalizing to β-actin or GAPDH mRNA inparallel TaqMan reactions. For each gene of interest an upper and lowerprimer and a fluorescently labeled probe are designed. Real timeincorporation of SYBR Green I dye into a specific PCR product can bemeasured in glass capillary tubes using a lightcyler. A standard curveis generated for each primer pair using control cRNA. Values arerepresented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Models Useful to Evaluate the Down-Regulation of BACE GeneExpression Cell Culture

Vassar et al., 1999, Science, 286, 735-741, describe a cell culturemodel for studying BACE inhibition. Specific antisense nucleic acidmolecules targeting BACE mRNA were used for inhibition studies ofendogenous BACE expression in 101 cells and APPsw (Swedish type amyloidprecursor protein expressing) cells via lipid mediated transfection.Antisense treatment resulted in dramatic reduction of both BACE mRNA byNorthern blot analysis, and APPsβsw (“Swedish” type β-secretase cleavageproduct) by ELISA, with maximum inhibition of both parameters at 75-80%.This model was also used to study the effect of BACE inhibition onamyloid β-peptide production in APPsw cells. Similarly, such a model canbe used to screen siRNA molecules of the instant invention for efficacyand potency.

In several cell culture systems, cationic lipids have been shown toenhance the bioavailability of oligonucleotides to cells in culture(Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In oneembodiment, siNA molecules of the invention are complexed with cationiclipids for cell culture experiments. siNA and cationic lipid mixturesare prepared in serum-free DMEM immediately prior to addition to thecells. DMEM plus additives are warmed to room temperature (about 20-25°C.) and cationic lipid is added to the final desired concentration andthe solution is vortexed briefly. siNA molecules are added to the finaldesired concentration and the solution is again vortexed briefly andincubated for 10 minutes at room temperature. In dose responseexperiments, the RNA/lipid complex is serially diluted into DMEMfollowing the 10 minute incubation.

Animal Models

Evaluating the efficacy of anti-BACE agents in animal models is animportant prerequisite to human clinical trials. Games et al., 1995,Nature, 373, 523-527, describe a transgenic mouse model in which mutanthuman familial type APP (Phe 717 instead of Val) is overexpressed. Thismodel results in mice that progressively develop many of thepathological hallmarks of Alzheimer's disease, and as such, provides amodel for testing therapeutic drugs, including siNA constructs of theinstant invention.

Example 9 RNAi Mediated Inhibition of BACE RNA Expression

siNA constructs (Tables II and III) are tested for efficacy in reducingBACE RNA expression in, for example in A549 cells. Cells are platedapproximately 24h before transfection in 96-well plates at 5,000-7,500cells/well, 100 μl/well, such that at the time of transfection cells are70-90% confluent. For transfection, annealed siNAs are mixed with thetransfection reagent (Lipofectamine 2000, Invitrogen) in a volume of 50μl/well and incubated for 20 min. at room temperature. The siNAtransfection mixtures are added to cells to give a final siNAconcentration of 25 nM in a volume of 150 μl. Each siNA transfectionmixture is added to 3 wells for triplicate siNA treatments. Cells areincubated at 37° for 24 hours in the continued presence of the siNAtransfection mixture. At 24 hours, RNA is prepared from each well oftreated cells. The supernatants with the transfection mixtures are firstremoved and discarded, then the cells are lysed and RNA prepared fromeach well. Target gene expression following treatment is evaluated byRT-PCR for the target gene and for a control gene (36B4, an RNApolymerase subunit) for normalization. The triplicate data is averagedand the standard deviations determined for each treatment. Normalizeddata are graphed and the percent reduction of target mRNA by activesiNAs in comparison to their respective inverted control siNAs wasdetermined.

In a non-limiting example, siNA constructs were screened for activity(see FIG. 12) and compared to untreated cells, scrambled siNA controlconstructs (Scram1 and Scram2), and cells transfected with lipid alone(transfection control). As shown in FIG. 12, the siNA constructs showsignificant reduction of BACE RNA expression. Leads generated from sucha screen are then further assayed. In a non-limiting example, siNAconstructs comprising ribonucleotides and 3′-terminal dithymidine capsare assayed along with a chemically modified siNA construct comprising2′-deoxy-2′-fluoro pyrimidine nucleotides and purine ribonucleotides, inwhich the sense strand of the siNA is further modified with 5′ and3′-terminal inverted deoxyabasic caps and the antisense strand comprisesa 3′-terminal phosphorothioate internucleotide linkage. Additionalstabilization chemistries as described in Table IV are similarly assayedfor activity. These siNA constructs are compared to appropriate matchedchemistry inverted controls. In addition, the siNA constructs are alsocompared to untreated cells, cells transfected with lipid and scrambledsiNA constructs, and cells transfected with lipid alone (transfectioncontrol). Results are shown in FIG. 13. A549 cells were transfected with0.25 ug/well of lipid complexed with 25 nM siNA. A lead siNA construct(31007/31083) chosen from the screen described in FIG. 12 above wasfurther modified using chemical modifications described in Table IVherein. Chemically modified constructs having Stab 4/5 chemistry(31378/31381) and Stab 7/11 chemistry (31384/31387) (see Table IV) weretested for efficacy compared to matched chemistry inverted controls(sequences of the siNA constructs are shown in Table III). The originallead siNA construct (31007/31083) and the Stab 4/5 and Stab 7/11constructs were compared to untreated cells, scrambled siNA controlconstructs (Scram1 and Scram2), and cells transfected with lipid alone(transfection control). As shown in FIG. 13, the original lead constructand the Stab 4/5 and Stab 7/11 modified siNA constructs all showsignificant reduction of BACE RNA expression.

Example 10 Indications

Particular degenerative and disease states that can be associated withBACE, APP, PIN-1, PS-1 and/or PS-2 expression modulation include but arenot limited to: Alzheimer's disease, dementia, stroke (CVA) and anyother diseases or conditions that are related to the levels of BACE,APP, PIN-1, PS-1 and/or PS-2 in a cell or tissue, alone or incombination with other therapies. The reduction of BACE, APP, PIN-1,PS-1 and/or PS-2 expression (specifically BACE, APP, PIN-1, PS-1 and/orPS-2 RNA levels) and thus reduction in the level of the respectiveprotein relieves, to some extent, the symptoms of the disease orcondition.

Those skilled in the art will recognize that other drug compounds andtherapies may be readily combined with or used in conjunction with thenucleic acid molecules of the instant invention (e.g., siNA molecules)are hence within the scope of the instant invention.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with an siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I BACE Accession Numbers NM_012104 Homo sapiens beta-siteAPP-cleaving enzyme (BACE), transcript variant a, mRNAgi|21040369|ref|NM_012104.2|[21040369] NM_006222 Homo sapiens protein(peptidyl-prolyl cis/trans isomerase) NIMA-interacting 1-like (PIN1L),mRNA gi|5453899|ref|NM_006222.1|[5453899] L76517 Homo sapiens (clonecc44) senilin 1 (PS1; S182) mRNA, complete cdsgi|1479973|gb|L76517.1|HUMPS1MRNA[1479973] L43964 Homo sapiens (cloneF-T03796) STM-2 mRNA, complete cdsgi|951202|gb|L43964.1|HUMSTM2R[951202] NM_138973 Homo sapiens beta-siteAPP-cleaving enzyme (BACE), transcript variant d, mRNAgi|21040367|ref|NM_138973.1|[21040367] NM_138972 Homo sapiens beta-siteAPP-cleaving enzyme (BACE), transcript variant b, mRNAgi|21040365|ref|NM_138972.1|[21040365] NM_138971 Homo sapiens beta-siteAPP-cleaving enzyme (BACE), transcript variant c, mRNAgi|21040363|ref|NM_138971.1|[21040363] AK075049 Homo sapiens cDNAFLJ90568 fis, clone OVARC1001570, highly similar to Homo sapiensbeta-site APP cleaving enzyme (BACE) mRNAgi|22760888|dbj|AK075049.1|[22760888] AF527782 Homo sapiens beta-siteAPP-cleaving enzyme (BACE) mRNA, partial cds, alternatively splicedgi|22094870|gb|AF527782.1|[22094870] AF324837 Homo sapiens beta-site APPcleaving enzyme mRNA, partial cds, alternatively splicedgi|21449275|gb|AF324837.1|[21449275] AF338817 Homo sapiens beta-site APPcleaving enzyme type C mRNA, complete cdsgi|13699247|gb|AF338817.1|[13699247] AF338816 Homo sapiens beta-site APPcleaving enzyme type B mRNA, complete cdsgi|13699245|gb|AF338816.1|[13699245] AB050438 Homo sapiens BACE mRNA forbeta-site APP cleaving enzyme I-432, complete cdsgi|13568410|dbj|AB050438.1|[13568410] AB050437 Homo sapiens BACE mRNAfor beta-site APP cleaving enzyme I-457, complete cdsgi|13568408|dbj|AB050437.1|[13568408] AB050436 Homo sapiens BACE mRNAfor beta-site APP cleaving enzyme I-476, complete cdsgi|13568406|dbj|AB050436.1|[13568406] AF190725 Homo sapiens beta-siteAPP cleaving enzyme (BACE) mRNA, complete cdsgi|6118538|gb|AF190725.1|AF190725[6118538] NM_007319 Homo sapienspresenilin 1 (Alzheimer disease 3) (PSEN1), transcript variant I-374.,mRNA gi|7549814|ref|NM_007319.1|[7549814] NM_138992 Homo sapiensbeta-site APP-cleaving enzyme 2 (BACE2), transcript variant b, mRNAgi|21040361|ref|NM_138992.1|[21040361] NM_138991 Homo sapiens beta-siteAPP-cleaving enzyme 2 (BACE2), transcript variant c, mRNAgi|21040359|ref|NM_138991.1|[21040359] NM_012105 Homo sapiens beta-siteAPP-cleaving enzyme 2 (BACE2), transcript variant a, mRNAgi|21040358|ref|NM_012105.3|[21040358] AB066441 Homo sapiens APP mRNAfor amyloid precursor protein, partial cds, D678N mutantgi|16904654|dbj|AB066441.1|[16904654] AB050438 Homo sapiens BACE mRNAfor beta-site APP cleaving enzyme I-432, complete cdsgi|13568410|dbj|AB050438.1|[13568410] AB050437 Homo sapiens BACE mRNAfor beta-site APP cleaving enzyme I-457, complete cdsgi|13568408|dbj|AB050437.1|[13568408] AB050436 Homo sapiens BACE mRNAfor beta-site APP cleaving enzyme I-476, complete cdsgi|13568406|dbj|AB050436.1|[13568406] NM_012486 Homo sapiens presenilin2 (Alzheimer disease 4) (PSEN2), transcript variant 2, mRNAgi|7108359|ref|NM_012486.1|[7108359] NM_000447 Homo sapiens presenilin 2(Alzheimer disease 4) (PSEN2), transcript variant 1, mRNAgi|4506164|ref|NM_000447.1|[4506164] AF188277 Homo sapiens aspartylprotease (BACE2) mRNA, complete cds, alternatively splicedgi|7025334|gb|AF188277.1|AF188277[7025334] AF188276 Homo sapiensaspartyl protease (BACE2) mRNA, complete cds, alternatively splicedgi|7025332|gb|AF188276.1|AF188276[7025332] AF178532 Homo sapiensaspartyl protease (BACE2) mRNA, complete cdsgi|6851265|gb|AF178532.1|AF178532[6851265] D87675 Homo sapiens DNA foramyloid precursor protein, complete cdsgi|2429080|dbj|D87675.1|[2429080] AF201468 Homo sapiens APPbeta-secretase mRNA, complete cdsgi|6601444|gb|AF201468.1|AF201468[6601444] AF190725 Homo sapiensbeta-site APP cleaving enzyme (BACE) mRNA, complete cdsgi|6118538|gb|AF190725.1|AF190725[6118538] E14707 DNA encoding a mutatedamyloid precursor proteingi|5709390|dbj|E14707.1||pat|JP|1998001499|1[5709390] AF168956 Homosapiens amyloid precursor protein homolog HSD-2 mRNA, complete cdsgi|5702387|gb|AF168956.1|AF168956[5702387] S60099 APPH = amyloidprecursor protein homolog [human, placenta, mRNA, 3727 nt]gi|300168|bbm|300685|bbs|131198|gb|S60099.1|S60099[300168] U50939 Humanamyloid precursor protein-binding protein 1 mRNA, complete cdsgi|1314559|gb|U50939.1|HSU50939[1314559]

TABLE II BACE siNA and Target Sequences NM_012104|BACE Seq Seq Seq PosTarget Sequence ID UPos Upper seq ID LPos Lower seq ID    1CGCACUCGUCCCCAGCCCG   1    1 CGCACUCGUCCCCAGCCCG   1   23CGGGCUGGGGACGAGUGCG 326   19 GCCCGGGAGCUGCGAGCCG   2   19GCCCGGGAGCUGCGAGCCG   2   41 CGGCUCGCAGCUCCCGGGC 327   37GCGAGCUGGAUUAUGGUGG   3   37 GCGAGCUGGAUUAUGGUGG   3   59CCACCAUAAUCCAGCUCGC 328   55 GCCUGAGCAGCCAACGCAG   4   55GCCUGAGCAGCCAACGCAG   4   77 CUGCGUUGGCUGCUCAGGC 329   73GCCGCAGGAGCCCGGAGCC   5   73 GCCGCAGGAGCCCGGAGCC   5   95GGCUCCGGGCUCCUGCGGC 330   91 CCUUGCCCCUGCCCGCGCC   6   91CCUUGCCCCUGCCCGCGCC   6  113 GGCGCGGGCAGGGGCAAGG 331  109CGCCGCCCGCCGGGGGGAC   7  109 CGCCGCCCGCCGGGGGGAC   7  131GUCCCCCCGGCGGGCGGCG 332  127 CCAGGGAAGCCGCCACCGG   8  127CCAGGGAAGCCGCCACCGG   8  149 CCGGUGGCGGCUUCCCUGG 333  145GCCCGCCAUGCCCGCCCCU   9  145 GCCCGCCAUGCCCGCCCCU   9  167AGGGGCGGGCAUGGCGGGC 334  163 UCCCAGCCCCGCCGGGAGC  10  163UCCCAGCCCCGCCGGGAGC  10  185 GCUCCCGGCGGGGCUGGGA 335  181CCCGCGCCCGCUGCCCAGG  11  181 CCCGCGCCCGCUGCCCAGG  11  203CCUGGGCAGCGGGCGCGGG 336  199 GCUGGCCGCCGCCGUGCCG  12  199GCUGGCCGCCGCCGUGCCG  12  221 CGGCACGGCGGCGGCCAGC 337  217GAUGUAGCGGGCUCCGGAU  13  217 GAUGUAGCGGGCUCCGGAU  13  239AUCCGGAGCCCGCUACAUC 338  235 UCCCAGCCUCUCCCCUGCU  14  235UCCCAGCCUCUCCCCUGCU  14  257 AGCAGGGGAGAGGCUGGGA 339  253UCCCGUGCUCUGCGGAUCU  15  253 UCCCGUGCUCUGCGGAUCU  15  275AGAUCCGCAGAGCACGGGA 340  271 UCCCCUGACCGCUCUCCAC  16  271UCCCCUGACCGCUCUCCAC  16  293 GUGGAGAGCGGUCAGGGGA 341  289CAGCCCGGACCCGGGGGCU  17  289 CAGCCCGGACCCGGGGGCU  17  311AGCCCCCGGGUCCGGGCUG 342  307 UGGCCCAGGGCCCUGCAGG  18  307UGGCCCAGGGCCCUGCAGG  18  329 CCUGCAGGGCCCUGGGCCA 343  325GCCCUGGCGUCCUGAUGCC  19  325 GCCCUGGCGUCCUGAUGCC  19  347GGCAUCAGGACGCCAGGGC 344  343 CCCCAAGCUCCCUCUCCUG  20  343CCCCAAGCUCCCUCUCCUG  20  365 CAGGAGAGGGAGCUUGGGG 345  361GAGAAGCCACCAGCACCAC  21  361 GAGAAGCCACCAGCACCAC  21  383GUGGUGCUGGUGGCUUCUC 346  379 CCCAGACUUGGGGGCAGGC  22  379CCCAGACUUGGGGGCAGGC  22  401 GCCUGCCCCCAAGUCUGGG 347  397CGCCAGGGACGGACGUGGG  23  397 CGCCAGGGACGGACGUGGG  23  419CCCACGUCCGUCCCUGGCG 348  415 GCCAGUGCGAGCCCAGAGG  24  415GCCAGUGCGAGCCCAGAGG  24  437 CCUCUGGGCUCGCACUGGC 349  433GGCCCGAAGGCCGGGGCCC  25  433 GGCCCGAAGGCCGGGGCCC  25  455GGGCCCCGGCCUUCGGGCC 350  451 CACCAUGGCCCAAGCCCUG  26  451CACCAUGGCCCAAGCCCUG  26  473 CAGGGCUUGGGCCAUGGUG 351  469GCCCUGGCUCCUGCUGUGG  27  469 GCCCUGGCUCCUGCUGUGG  27  491CCACAGCAGGAGCCAGGGC 352  487 GAUGGGCGCGGGAGUGCUG  28  487GAUGGGCGCGGGAGUGCUG  28  509 CAGCACUCCCGCGCCCAUC 353  505GCCUGCCCACGGCACCCAG  29  505 GCCUGCCCACGGCACCCAG  29  527CUGGGUGCCGUGGGCAGGC 354  523 GCACGGCAUCCGGCUGCCC  30  523GCACGGCAUCCGGCUGCCC  30  545 GGGCAGCCGGAUGCCGUGC 355  541CCUGCGCAGCGGCCUGGGG  31  541 CCUGCGCAGCGGCCUGGGG  31  563CCCCAGGCCGCUGCGCAGG 356  559 GGGCGCCCCCCUGGGGCUG  32  559GGGCGCCCCCCUGGGGCUG  32  581 CAGCCCCAGGGGGGCGCCC 357  577GCGGCUGCCCCGGGAGACC  33  577 GCGGCUGCCCCGGGAGACC  33  599GGUCUCCCGGGGCAGCCGC 358  595 CGACGAAGAGCCCGAGGAG  34  595CGACGAAGAGCCCGAGGAG  34  617 CUCCUCGGGCUCUUCGUCG 359  613GCCCGGCCGGAGGGGCAGC  35  613 GCCCGGCCGGAGGGGCAGC  35  635GCUGCCCCUCCGGCCGGGC 360  631 CUUUGUGGAGAUGGUGGAC  36  631CUUUGUGGAGAUGGUGGAC  36  653 GUCCACCAUCUCCACAAAG 361  649CAACCUGAGGGGCAAGUCG  37  649 CAACCUGAGGGGCAAGUCG  37  671CGACUUGCCCCUCAGGUUG 362  667 GGGGCAGGGCUACUACGUG  38  667GGGGCAGGGCUACUACGUG  38  689 CACGUAGUAGCCCUGCCCC 363  685GGAGAUGACCGUGGGCAGC  39  685 GGAGAUGACCGUGGGCAGC  39  707GCUGCCCACGGUCAUCUCC 364  703 CCCCCCGCAGACGCUCAAC  40  703CCCCCCGCAGACGCUCAAC  40  725 GUUGAGCGUCUGCGGGGGG 365  721CAUCCUGGUGGAUACAGGC  41  721 CAUCCUGGUGGAUACAGGC  41  743GCCUGUAUCCACCAGGAUG 366  739 CAGCAGUAACUUUGCAGUG  42  739CAGCAGUAACUUUGCAGUG  42  761 CACUGCAAAGUUACUGCUG 367  757GGGUGCUGCCCCCCACCCC  43  757 GGGUGCUGCCCCCCACCCC  43  779GGGGUGGGGGGCAGCACCC 368  775 CUUCCUGCAUCGCUACUAC  44  775CUUCCUGCAUCGCUACUAC  44  797 GUAGUAGCGAUGCAGGAAG 369  793CCAGAGGCAGCUGUCCAGC  45  793 CCAGAGGCAGCUGUCCAGC  45  815GCUGGACAGCUGCCUCUGG 370  811 CACAUACCGGGACCUCCGG  46  811CACAUACCGGGACCUCCGG  46  833 CCGGAGGUCCCGGUAUGUG 371  829GAAGGGUGUGUAUGUGCCC  47  829 GAAGGGUGUGUAUGUGCCC  47  851GGGCACAUACACACCCUUC 372  847 CUACACCCAGGGCAAGUGG  48  847CUACACCCAGGGCAAGUGG  48  869 CCACUUGCCCUGGGUGUAG 373  865GGAAGGGGAGCUGGGCACC  49  865 GGAAGGGGAGCUGGGCACC  49  887GGUGCCCAGCUCCCCUUCC 374  883 CGACCUGGUAAGCAUCCCC  50  883CGACCUGGUAAGCAUCCCC  50  905 GGGGAUGCUUACCAGGUCG 375  901CCAUGGCCCCAACGUCACU  51  901 CCAUGGCCCCAACGUCACU  51  923AGUGACGUUGGGGCCAUGG 376  919 UGUGCGUGCCAACAUUGCU  52  919UGUGCGUGCCAACAUUGCU  52  941 AGCAAUGUUGGCACGCACA 377  937UGCCAUCACUGAAUCAGAC  53  937 UGCCAUCACUGAAUCAGAC  53  959GUCUGAUUCAGUGAUGGCA 378  955 CAAGUUCUUCAUCAACGGC  54  955CAAGUUCUUCAUCAACGGC  54  977 GCCGUUGAUGAAGAACUUG 379  973CUCCAACUGGGAAGGCAUC  55  973 CUCCAACUGGGAAGGCAUC  55  995GAUGCCUUCCCAGUUGGAG 380  991 CCUGGGGCUGGCCUAUGCU  56  991CCUGGGGCUGGCCUAUGCU  56 1013 AGCAUAGGCCAGCCCCAGG 381 1009UGAGAUUGCCAGGCCUGAC  57 1009 UGAGAUUGCCAGGCCUGAC  57 1031GUCAGGCCUGGCAAUCUCA 382 1027 CGACUCCCUGGAGCCUUUC  58 1027CGACUCCCUGGAGCCUUUC  58 1049 GAAAGGCUCCAGGGAGUCG 383 1045CUUUGACUCUCUGGUAAAG  59 1045 CUUUGACUCUCUGGUAAAG  59 1067CUUUACCAGAGAGUCAAAG 384 1063 GCAGACCCACGUUCCCAAC  60 1063GCAGACCCACGUUCCCAAC  60 1085 GUUGGGAACGUGGGUCUGC 385 1081CCUCUUCUCCCUGCAGCUU  61 1081 CCUCUUCUCCCUGCAGCUU  61 1103AAGCUGCAGGGAGAAGAGG 386 1099 UUGUGGUGCUGGCUUCCCC  62 1099UUGUGGUGCUGGCUUCCCC  62 1121 GGGGAAGCCAGCACCACAA 387 1117CCUCAACCAGUCUGAAGUG  63 1117 CCUCAACCAGUCUGAAGUG  63 1139CACUUCAGACUGGUUGAGG 388 1135 GCUGGCCUCUGUCGGAGGG  64 1135GCUGGCCUCUGUCGGAGGG  64 1157 CCCUCCGACAGAGGCCAGC 389 1153GAGCAUGAUCAUUGGAGGU  65 1153 GAGCAUGAUCAUUGGAGGU  65 1175ACCUCCAAUGAUCAUGCUC 390 1171 UAUCGACCACUCGCUGUAC  66 1171UAUCGACCACUCGCUGUAC  66 1193 GUACAGCGAGUGGUCGAUA 391 1189CACAGGCAGUCUCUGGUAU  67 1189 CACAGGCAGUCUCUGGUAU  67 1211AUACCAGAGACUGCCUGUG 392 1207 UACACCCAUCCGGCGGGAG  68 1207UACACCCAUCCGGCGGGAG  68 1229 CUCCCGCCGGAUGGGUGUA 393 1225GUGGUAUUAUGAGGUCAUC  69 1225 GUGGUAUUAUGAGGUCAUC  69 1247GAUGACCUCAUAAUACCAC 394 1243 CAUUGUGCGGGUGGAGAUC  70 1243CAUUGUGCGGGUGGAGAUC  70 1265 GAUCUCCACCCGCACAAUG 395 1261CAAUGGACAGGAUCUGAAA  71 1261 CAAUGGACAGGAUCUGAAA  71 1283UUUCAGAUCCUGUCCAUUG 396 1279 AAUGGACUGCAAGGAGUAC  72 1279AAUGGACUGCAAGGAGUAC  72 1301 GUACUCCUUGCAGUCCAUU 397 1297CAACUAUGACAAGAGCAUU  73 1297 CAACUAUGACAAGAGCAUU  73 1319AAUGCUCUUGUCAUAGUUG 398 1315 UGUGGACAGUGGCACCACC  74 1315UGUGGACAGUGGCACCACC  74 1337 GGUGGUGCCACUGUCCACA 399 1333CAACCUUCGUUUGCCCAAG  75 1333 CAACCUUCGUUUGCCCAAG  75 1355CUUGGGCAAACGAAGGUUG 400 1351 GAAAGUGUUUGAAGCUGCA  76 1351GAAAGUGUUUGAAGCUGCA  76 1373 UGCAGCUUCAAACACUUUC 401 1369AGUCAAAUCCAUCAAGGCA  77 1369 AGUCAAAUCCAUCAAGGCA  77 1391UGCCUUGAUGGAUUUGACU 402 1387 AGCCUCCUCCACGGAGAAG  78 1387AGCCUCCUCCACGGAGAAG  78 1409 CUUCUCCGUGGAGGAGGCU 403 1405GUUCCCUGAUGGUUUCUGG  79 1405 GUUCCCUGAUGGUUUCUGG  79 1427CCAGAAACCAUCAGGGAAC 404 1423 GCUAGGAGAGCAGCUGGUG  80 1423GCUAGGAGAGCAGCUGGUG  80 1445 CACCAGCUGCUCUCCUAGC 405 1441GUGCUGGCAAGCAGGCACC  81 1441 GUGCUGGCAAGCAGGCACC  81 1463GGUGCCUGCUUGCCAGCAC 406 1459 CACCCCUUGGAACAUUUUC  82 1459CACCCCUUGGAACAUUUUC  82 1481 GAAAAUGUUCCAAGGGGUG 407 1477CCCAGUCAUCUCACUCUAC  83 1477 CCCAGUCAUCUCACUCUAC  83 1499GUAGAGUGAGAUGACUGGG 408 1495 CCUAAUGGGUGAGGUUACC  84 1495CCUAAUGGGUGAGGUUACC  84 1517 GGUAACCUCACCCAUUAGG 409 1513CAACCAGUCCUUCCGCAUC  85 1513 CAACCAGUCCUUCCGCAUC  85 1535GAUGCGGAAGGACUGGUUG 410 1531 CACCAUCCUUCCGCAGCAA  86 1531CACCAUCCUUCCGCAGCAA  86 1553 UUGCUGCGGAAGGAUGGUG 411 1549AUACCUGCGGCCAGUGGAA  87 1549 AUACCUGCGGCCAGUGGAA  87 1571UUCCACUGGCCGCAGGUAU 412 1567 AGAUGUGGCCACGUCCCAA  88 1567AGAUGUGGCCACGUCCCAA  88 1589 UUGGGACGUGGCCACAUCU 413 1585AGACGACUGUUACAAGUUU  89 1585 AGACGACUGUUACAAGUUU  89 1607AAACUUGUAACAGUCGUCU 414 1603 UGCCAUCUCACAGUCAUCC  90 1603UGCCAUCUCACAGUCAUCC  90 1625 GGAUGACUGUGAGAUGGCA 415 1621CACGGGCACUGUUAUGGGA  91 1621 CACGGGCACUGUUAUGGGA  91 1643UCCCAUAACAGUGCCCGUG 416 1639 AGCUGUUAUCAUGGAGGGC  92 1639AGCUGUUAUCAUGGAGGGC  92 1661 GCCCUCCAUGAUAACAGCU 417 1657CUUCUACGUUGUCUUUGAU  93 1657 CUUCUACGUUGUCUUUGAU  93 1679AUCAAAGACAACGUAGAAG 418 1675 UCGGGCCCGAAAACGAAUU  94 1675UCGGGCCCGAAAACGAAUU  94 1697 AAUUCGUUUUCGGGCCCGA 419 1693UGGCUUUGCUGUCAGCGCU  95 1693 UGGCUUUGCUGUCAGCGCU  95 1715AGCGCUGACAGCAAAGCCA 420 1711 UUGCCAUGUGCACGAUGAG  96 1711UUGCCAUGUGCACGAUGAG  96 1733 CUCAUCGUGCACAUGGCAA 421 1729GUUCAGGACGGCAGCGGUG  97 1729 GUUCAGGACGGCAGCGGUG  97 1751CACCGCUGCCGUCCUGAAC 422 1747 GGAAGGCCCUUUUGUCACC  98 1747GGAAGGCCCUUUUGUCACC  98 1769 GGUGACAAAAGGGCCUUCC 423 1765CUUGGACAUGGAAGACUGU  99 1765 CUUGGACAUGGAAGACUGU  99 1787ACAGUCUUCCAUGUCCAAG 424 1783 UGGCUACAACAUUCCACAG 100 1783UGGCUACAACAUUCCACAG 100 1805 CUGUGGAAUGUUGUAGCCA 425 1801GACAGAUGAGUCAACCCUC 101 1801 GACAGAUGAGUCAACCCUC 101 1823GAGGGUUGACUCAUCUGUC 426 1819 CAUGACCAUAGCCUAUGUC 102 1819CAUGACCAUAGCCUAUGUC 102 1841 GACAUAGGCUAUGGUCAUG 427 1837CAUGGCUGCCAUCUGCGCC 103 1837 CAUGGCUGCCAUCUGCGCC 103 1859GGCGCAGAUGGCAGCCAUG 428 1855 CCUCUUCAUGCUGCCACUC 104 1855CCUCUUCAUGCUGCCACUC 104 1877 GAGUGGCAGCAUGAAGAGG 429 1873CUGCCUCAUGGUGUGUCAG 105 1873 CUGCCUCAUGGUGUGUCAG 105 1895CUGACACACCAUGAGGCAG 430 1891 GUGGCGCUGCCUCCGCUGC 106 1891GUGGCGCUGCCUCCGCUGC 106 1913 GCAGCGGAGGCAGCGCCAC 431 1909CCUGCGCCAGCAGCAUGAU 107 1909 CCUGCGCCAGCAGCAUGAU 107 1931AUCAUGCUGCUGGCGCAGG 432 1927 UGACUUUGCUGAUGACAUC 108 1927UGACUUUGCUGAUGACAUC 108 1949 GAUGUCAUCAGCAAAGUCA 433 1945CUCCCUGCUGAAGUGAGGA 109 1945 CUCCCUGCUGAAGUGAGGA 109 1967UCCUCACUUCAGCAGGGAG 434 1963 AGGCCCAUGGGCAGAAGAU 110 1963AGGCCCAUGGGCAGAAGAU 110 1985 AUCUUCUGCCCAUGGGCCU 435 1981UAGAGAUUCCCCUGGACCA 111 1981 UAGAGAUUCCCCUGGACCA 111 2003UGGUCCAGGGGAAUCUCUA 436 1999 ACACCUCCGUGGUUCACUU 112 1999ACACCUCCGUGGUUCACUU 112 2021 AAGUGAACCACGGAGGUGU 437 2017UUGGUCACAAGUAGGAGAC 113 2017 UUGGUCACAAGUAGGAGAC 113 2039GUCUCCUACUUGUGACCAA 438 2035 CACAGAUGGCACCUGUGGC 114 2035CACAGAUGGCACCUGUGGC 114 2057 GCCACAGGUGCCAUCUGUG 439 2053CCAGAGCACCUCAGGACCC 115 2053 CCAGAGCACCUCAGGACCC 115 2075GGGUCCUGAGGUGCUCUGG 440 2071 CUCCCCACCCACCAAAUGC 116 2071CUCCCCACCCACCAAAUGC 116 2093 GCAUUUGGUGGGUGGGGAG 441 2089CCUCUGCCUUGAUGGAGAA 117 2089 CCUCUGCCUUGAUGGAGAA 117 2111UUCUCCAUCAAGGCAGAGG 442 2107 AGGAAAAGGCUGGCAAGGU 118 2107AGGAAAAGGCUGGCAAGGU 118 2129 ACCUUGCCAGCCUUUUCCU 443 2125UGGGUUCCAGGGACUGUAC 119 2125 UGGGUUCCAGGGACUGUAC 119 2147GUACAGUCCCUGGAACCCA 444 2143 CCUGUAGGAAACAGAAAAG 120 2143CCUGUAGGAAACAGAAAAG 120 2165 CUUUUCUGUUUCCUACAGG 445 2161GAGAAGAAAGAAGCACUCU 121 2161 GAGAAGAAAGAAGCACUCU 121 2183AGAGUGCUUCUUUCUUCUC 446 2179 UGCUGGCGGGAAUACUCUU 122 2179UGCUGGCGGGAAUACUCUU 122 2201 AAGAGUAUUCCCGCCAGCA 447 2197UGGUCACCUCAAAUUUAAG 123 2197 UGGUCACCUCAAAUUUAAG 123 2219CUUAAAUUUGAGGUGACCA 448 2215 GUCGGGAAAUUCUGCUGCU 124 2215GUCGGGAAAUUCUGCUGCU 124 2237 AGCAGCAGAAUUUCCCGAC 449 2233UUGAAACUUCAGCCCUGAA 125 2233 UUGAAACUUCAGCCCUGAA 125 2255UUCAGGGCUGAAGUUUCAA 450 2251 ACCUUUGUCCACCAUUCCU 126 2251ACCUUUGUCCACCAUUCCU 126 2273 AGGAAUGGUGGACAAAGGU 451 2269UUUAAAUUCUCCAACCCAA 127 2269 UUUAAAUUCUCCAACCCAA 127 2291UUGGGUUGGAGAAUUUAAA 452 2287 AAGUAUUCUUCUUUUCUUA 128 2287AAGUAUUCUUCUUUUCUUA 128 2309 UAAGAAAAGAAGAAUACUU 453 2305AGUUUCAGAAGUACUGGCA 129 2305 AGUUUCAGAAGUACUGGCA 129 2327UGCCAGUACUUCUGAAACU 454 2323 AUCACACGCAGGUUACCUU 130 2323AUCACACGCAGGUUACCUU 130 2345 AAGGUAACCUGCGUGUGAU 455 2341UGGCGUGUGUCCCUGUGGU 131 2341 UGGCGUGUGUCCCUGUGGU 131 2363ACCACAGGGACACACGCCA 456 2359 UACCCUGGCAGAGAAGAGA 132 2359UACCCUGGCAGAGAAGAGA 132 2381 UCUCUUCUCUGCCAGGGUA 457 2377ACCAAGCUUGUUUCCCUGC 133 2377 ACCAAGCUUGUUUCCCUGC 133 2399GCAGGGAAACAAGCUUGGU 458 2395 CUGGCCAAAGUCAGUAGGA 134 2395CUGGCCAAAGUCAGUAGGA 134 2417 UCCUACUGACUUUGGCCAG 459 2413AGAGGAUGCACAGUUUGCU 135 2413 AGAGGAUGCACAGUUUGCU 135 2435AGCAAACUGUGCAUCCUCU 460 2431 UAUUUGCUUUAGAGACAGG 136 2431UAUUUGCUUUAGAGACAGG 136 2453 CCUGUCUCUAAAGCAAAUA 461 2449GGACUGUAUAAACAAGCCU 137 2449 GGACUGUAUAAACAAGCCU 137 2471AGGCUUGUUUAUACAGUCC 462 2467 UAACAUUGGUGCAAAGAUU 138 2467UAACAUUGGUGCAAAGAUU 138 2489 AAUCUUUGCACCAAUGUUA 463 2485UGCCUCUUGAAUUAAAAAA 139 2485 UGCCUCUUGAAUUAAAAAA 139 2507UUUUUUAAUUCAAGAGGCA 464 2503 AAAAAACUAGAUUGACUAU 140 2503AAAAAACUAGAUUGACUAU 140 2525 AUAGUCAAUCUAGUUUUUU 465 2521UUUAUACAAAUGGGGGCGG 141 2521 UUUAUACAAAUGGGGGCGG 141 2543CCGCCCCCAUUUGUAUAAA 466 2539 GCUGGAAAGAGGAGAAGGA 142 2539GCUGGAAAGAGGAGAAGGA 142 2561 UCCUUCUCCUCUUUCCAGC 467 2557AGAGGGAGUACAAAGACAG 143 2557 AGAGGGAGUACAAAGACAG 143 2579CUGUCUUUGUACUCCCUCU 468 2575 GGGAAUAGUGGGAUCAAAG 144 2575GGGAAUAGUGGGAUCAAAG 144 2597 CUUUGAUCCCACUAUUCCC 469 2593GCUAGGAAAGGCAGAAACA 145 2593 GCUAGGAAAGGCAGAAACA 145 2615UGUUUCUGCCUUUCCUAGC 470 2611 ACAACCACUCACCAGUCCU 146 2611ACAACCACUCACCAGUCCU 146 2633 AGGACUGGUGAGUGGUUGU 471 2629UAGUUUUAGACCUCAUCUC 147 2629 UAGUUUUAGACCUCAUCUC 147 2651GAGAUGAGGUCUAAAACUA 472 2647 CCAAGAUAGCAUCCCAUCU 148 2647CCAAGAUAGCAUCCCAUCU 148 2669 AGAUGGGAUGCUAUCUUGG 473 2665UCAGAAGAUGGGUGUUGUU 149 2665 UCAGAAGAUGGGUGUUGUU 149 2687AACAACACCCAUCUUCUGA 474 2683 UUUCAAUGUUUUCUUUUCU 150 2683UUUCAAUGUUUUCUUUUCU 150 2705 AGAAAAGAAAACAUUGAAA 475 2701UGUGGUUGCAGCCUGACCA 151 2701 UGUGGUUGCAGCCUGACCA 151 2723UGGUCAGGCUGCAACCACA 476 2719 AAAAGUGAGAUGGGAAGGG 152 2719AAAAGUGAGAUGGGAAGGG 152 2741 CCCUUCCCAUCUCACUUUU 477 2737GCUUAUCUAGCCAAAGAGC 153 2737 GCUUAUCUAGCCAAAGAGC 153 2759GCUCUUUGGCUAGAUAAGC 478 2755 CUCUUUUUUAGCUCUCUUA 154 2755CUCUUUUUUAGCUCUCUUA 154 2777 UAAGAGAGCUAAAAAAGAG 479 2773AAAUGAAGUGCCCACUAAG 155 2773 AAAUGAAGUGCCCACUAAG 155 2795CUUAGUGGGCACUUCAUUU 480 2791 GAAGUUCCACUUAACACAU 156 2791GAAGUUCCACUUAACACAU 156 2813 AUGUGUUAAGUGGAACUUC 481 2809UGAAUUUCUGCCAUAUUAA 157 2809 UGAAUUUCUGCCAUAUUAA 157 2831UUAAUAUGGCAGAAAUUCA 482 2827 AUUUCAUUGUCUCUAUCUG 158 2827AUUUCAUUGUCUCUAUCUG 158 2849 CAGAUAGAGACAAUGAAAU 483 2845GAACCACCCUUUAUUCUAC 159 2845 GAACCACCCUUUAUUCUAC 159 2867GUAGAAUAAAGGGUGGUUC 484 2863 CAUAUGAUAGGCAGCACUG 160 2863CAUAUGAUAGGCAGCACUG 160 2885 CAGUGCUGCCUAUCAUAUG 485 2881GAAAUAUCCUAACCCCCUA 161 2881 GAAAUAUCCUAACCCCCUA 161 2903UAGGGGGUUAGGAUAUUUC 486 2899 AAGCUCCAGGUGCCCUGUG 162 2899AAGCUCCAGGUGCCCUGUG 162 2921 CACAGGGCACCUGGAGCUU 487 2917GGGAGAGCAACUGGACUAU 163 2917 GGGAGAGCAACUGGACUAU 163 2939AUAGUCCAGUUGCUCUCCC 488 2935 UAGCAGGGCUGGGCUCUGU 164 2935UAGCAGGGCUGGGCUCUGU 164 2957 ACAGAGCCCAGCCCUGCUA 489 2953UCUUCCUGGUCAUAGGCUC 165 2953 UCUUCCUGGUCAUAGGCUC 165 2975GAGCCUAUGACCAGGAAGA 490 2971 CACUCUUUCCCCCAAAUCU 166 2971CACUCUUUCCCCCAAAUCU 166 2993 AGAUUUGGGGGAAAGAGUG 491 2989UUCCUCUGGAGCUUUGCAG 167 2989 UUCCUCUGGAGCUUUGCAG 167 3011CUGCAAAGCUCCAGAGGAA 492 3007 GCCAAGGUGCUAAAAGGAA 168 3007GCCAAGGUGCUAAAAGGAA 168 3029 UUCCUUUUAGCACCUUGGC 493 3025AUAGGUAGGAGACCUCUUC 169 3025 AUAGGUAGGAGACCUCUUC 169 3047GAAGAGGUCUCCUACCUAU 494 3043 CUAUCUAAUCCUUAAAAGC 170 3043CUAUCUAAUCCUUAAAAGC 170 3065 GCUUUUAAGGAUUAGAUAG 495 3061CAUAAUGUUGAACAUUCAU 171 3061 CAUAAUGUUGAACAUUCAU 171 3083AUGAAUGUUCAACAUUAUG 496 3079 UUCAACAGCUGAUGCCCUA 172 3079UUCAACAGCUGAUGCCCUA 172 3101 UAGGGCAUCAGCUGUUGAA 497 3097AUAACCCCUGCCUGGAUUU 173 3097 AUAACCCCUGCCUGGAUUU 173 3119AAAUCCAGGCAGGGGUUAU 498 3115 UCUUCCUAUUAGGCUAUAA 174 3115UCUUCCUAUUAGGCUAUAA 174 3137 UUAUAGCCUAAUAGGAAGA 499 3133AGAAGUAGCAAGAUCUUUA 175 3133 AGAAGUAGCAAGAUCUUUA 175 3155UAAAGAUCUUGCUACUUCU 500 3151 ACAUAAUUCAGAGUGGUUU 176 3151ACAUAAUUCAGAGUGGUUU 176 3173 AAACCACUCUGAAUUAUGU 501 3169UCAUUGCCUUCCUACCCUC 177 3169 UCAUUGCCUUCCUACCCUC 177 3191GAGGGUAGGAAGGCAAUGA 502 3187 CUCUAAUGGCCCCUCCAUU 178 3187CUCUAAUGGCCCCUCCAUU 178 3209 AAUGGAGGGGCCAUUAGAG 503 3205UUAUUUGACUAAAGCAUCA 179 3205 UUAUUUGACUAAAGCAUCA 179 3227UGAUGCUUUAGUCAAAUAA 504 3223 ACACAGUGGCACUAGCAUU 180 3223ACACAGUGGCACUAGCAUU 180 3245 AAUGCUAGUGCCACUGUGU 505 3241UAUACCAAGAGUAUGAGAA 181 3241 UAUACCAAGAGUAUGAGAA 181 3263UUCUCAUACUCUUGGUAUA 506 3259 AAUACAGUGCUUUAUGGCU 182 3259AAUACAGUGCUUUAUGGCU 182 3281 AGCCAUAAAGCACUGUAUU 507 3277UCUAACAUUACUGCCUUCA 183 3277 UCUAACAUUACUGCCUUCA 183 3299UGAAGGCAGUAAUGUUAGA 508 3295 AGUAUCAAGGCUGCCUGGA 184 3295AGUAUCAAGGCUGCCUGGA 184 3317 UCCAGGCAGCCUUGAUACU 509 3313AGAAAGGAUGGCAGCCUCA 185 3313 AGAAAGGAUGGCAGCCUCA 185 3335UGAGGCUGCCAUCCUUUCU 510 3331 AGGGCUUCCUUAUGUCCUC 186 3331AGGGCUUCCUUAUGUCCUC 186 3353 GAGGACAUAAGGAAGCCCU 511 3349CCACCACAAGAGCUCCUUG 187 3349 CCACCACAAGAGCUCCUUG 187 3371CAAGGAGCUCUUGUGGUGG 512 3367 GAUGAAGGUCAUCUUUUUC 188 3367GAUGAAGGUCAUCUUUUUC 188 3389 GAAAAAGAUGACCUUCAUC 513 3385CCCCUAUCCUGUUCUUCCC 189 3385 CCCCUAUCCUGUUCUUCCC 189 3407GGGAAGAACAGGAUAGGGG 514 3403 CCUCCCCGCUCCUAAUGGU 190 3403CCUCCCCGCUCCUAAUGGU 190 3425 ACCAUUAGGAGCGGGGAGG 515 3421UACGUGGGUACCCAGGCUG 191 3421 UACGUGGGUACCCAGGCUG 191 3443CAGCCUGGGUACCCACGUA 516 3439 GGUUCUUGGGCUAGGUAGU 192 3439GGUUCUUGGGCUAGGUAGU 192 3461 ACUACCUAGCCCAAGAACC 517 3457UGGGGACCAAGUUCAUUAC 193 3457 UGGGGACCAAGUUCAUUAC 193 3479GUAAUGAACUUGGUCCCCA 518 3475 CCUCCCUAUCAGUUCUAGC 194 3475CCUCCCUAUCAGUUCUAGC 194 3497 GCUAGAACUGAUAGGGAGG 519 3493CAUAGUAAACUACGGUACC 195 3493 CAUAGUAAACUACGGUACC 195 3515GGUACCGUAGUUUACUAUG 520 3511 CAGUGUUAGUGGGAAGAGC 196 3511CAGUGUUAGUGGGAAGAGC 196 3533 GCUCUUCCCACUAACACUG 521 3529CUGGGUUUUCCUAGUAUAC 197 3529 CUGGGUUUUCCUAGUAUAC 197 3551GUAUACUAGGAAAACCCAG 522 3547 CCCACUGCAUCCUACUCCU 198 3547CCCACUGCAUCCUACUCCU 198 3569 AGGAGUAGGAUGCAGUGGG 523 3565UACCUGGUCAACCCGCUGC 199 3565 UACCUGGUCAACCCGCUGC 199 3587GCAGCGGGUUGACCAGGUA 524 3583 CUUCCAGGUAUGGGACCUG 200 3583CUUCCAGGUAUGGGACCUG 200 3605 CAGGUCCCAUACCUGGAAG 525 3601GCUAAGUGUGGAAUUACCU 201 3601 GCUAAGUGUGGAAUUACCU 201 3623AGGUAAUUCCACACUUAGC 526 3619 UGAUAAGGGAGAGGGAAAU 202 3619UGAUAAGGGAGAGGGAAAU 202 3641 AUUUCCCUCUCCCUUAUCA 527 3637UACAAGGAGGGCCUCUGGU 203 3637 UACAAGGAGGGCCUCUGGU 203 3659ACCAGAGGCCCUCCUUGUA 528 3655 UGUUCCUGGCCUCAGCCAG 204 3655UGUUCCUGGCCUCAGCCAG 204 3677 CUGGCUGAGGCCAGGAACA 529 3673GCUGCCCACAAGCCAUAAA 205 3673 GCUGCCCACAAGCCAUAAA 205 3695UUUAUGGCUUGUGGGCAGC 530 3691 ACCAAUAAAACAAGAAUAC 206 3691ACCAAUAAAACAAGAAUAC 206 3713 GUAUUCUUGUUUUAUUGGU 531 3709CUGAGUCAGUUUUUUAUCU 207 3709 CUGAGUCAGUUUUUUAUCU 207 3731AGAUAAAAAACUGACUCAG 532 3727 UGGGUUCUCUUCAUUCCCA 208 3727UGGGUUCUCUUCAUUCCCA 208 3749 UGGGAAUGAAGAGAACCCA 533 3745ACUGCACUUGGUGCUGCUU 209 3745 ACUGCACUUGGUGCUGCUU 209 3767AAGCAGCACCAAGUGCAGU 534 3763 UUGGCUGACUGGGAACACC 210 3763UUGGCUGACUGGGAACACC 210 3785 GGUGUUCCCAGUCAGCCAA 535 3781CCCAUAACUACAGAGUCUG 211 3781 CCCAUAACUACAGAGUCUG 211 3803CAGACUCUGUAGUUAUGGG 536 3799 GACAGGAAGACUGGAGACU 212 3799GACAGGAAGACUGGAGACU 212 3821 AGUCUCCAGUCUUCCUGUC 537 3817UGUCCACUUCUAGCUCGGA 213 3817 UGUCCACUUCUAGCUCGGA 213 3839UCCGAGCUAGAAGUGGACA 538 3835 AACUUACUGUGUAAAUAAA 214 3835AACUUACUGUGUAAAUAAA 214 3857 UUUAUUUACACAGUAAGUU 539 3853ACUUUCAGAACUGCUACCA 215 3853 ACUUUCAGAACUGCUACCA 215 3875UGGUAGCAGUUCUGAAAGU 540 3871 AUGAAGUGAAAAUGCCACA 216 3871AUGAAGUGAAAAUGCCACA 216 3893 UGUGGCAUUUUCACUUCAU 541 3889AUUUUGCUUUAUAAUUUCU 217 3889 AUUUUGCUUUAUAAUUUCU 217 3911AGAAAUUAUAAAGCAAAAU 542 3907 UACCCAUGUUGGGAAAAAC 218 3907UACCCAUGUUGGGAAAAAC 218 3929 GUUUUUCCCAACAUGGGUA 543 3925CUGGCUUUUUCCCAGCCCU 219 3925 CUGGCUUUUUCCCAGCCCU 219 3947AGGGCUGGGAAAAAGCCAG 544 3943 UUUCCAGGGCAUAAAACUC 220 3943UUUCCAGGGCAUAAAACUC 220 3965 GAGUUUUAUGCCCUGGAAA 545 3961CAACCCCUUCGAUAGCAAG 221 3961 CAACCCCUUCGAUAGCAAG 221 3983CUUGCUAUCGAAGGGGUUG 546 3979 GUCCCAUCAGCCUAUUAUU 222 3979GUCCCAUCAGCCUAUUAUU 222 4001 AAUAAUAGGCUGAUGGGAC 547 3997UUUUUUAAAGAAAACUUGC 223 3997 UUUUUUAAAGAAAACUUGC 223 4019GCAAGUUUUCUUUAAAAAA 548 4015 CACUUGUUUUUCUUUUUAC 224 4015CACUUGUUUUUCUUUUUAC 224 4037 GUAAAAAGAAAAACAAGUG 549 4033CAGUUACUUCCUUCCUGCC 225 4033 CAGUUACUUCCUUCCUGCC 225 4055GGCAGGAAGGAAGUAACUG 550 4051 CCCAAAAUUAUAAACUCUA 226 4051CCCAAAAUUAUAAACUCUA 226 4073 UAGAGUUUAUAAUUUUGGG 551 4069AAGUGUAAAAAAAAGUCUU 227 4069 AAGUGUAAAAAAAAGUCUU 227 4091AAGACUUUUUUUUACACUU 552 4087 UAACAACAGCUUCUUGCUU 228 4087UAACAACAGCUUCUUGCUU 228 4109 AAGCAAGAAGCUGUUGUUA 553 4105UGUAAAAAUAUGUAUUAUA 229 4105 UGUAAAAAUAUGUAUUAUA 229 4127UAUAAUACAUAUUUUUACA 554 4123 ACAUCUGUAUUUUUAAAUU 230 4123ACAUCUGUAUUUUUAAAUU 230 4145 AAUUUAAAAAUACAGAUGU 555 4141UCUGCUCCUGAAAAAUGAC 231 4141 UCUGCUCCUGAAAAAUGAC 231 4163GUCAUUUUUCAGGAGCAGA 556 4159 CUGUCCCAUUCUCCACUCA 232 4159CUGUCCCAUUCUCCACUCA 232 4181 UGAGUGGAGAAUGGGACAG 557 4177ACUGCAUUUGGGGCCUUUC 233 4177 ACUGCAUUUGGGGCCUUUC 233 4199GAAAGGCCCCAAAUGCAGU 558 4195 CCCAUUGGUCUGCAUGUCU 234 4195CCCAUUGGUCUGCAUGUCU 234 4217 AGACAUGCAGACCAAUGGG 559 4213UUUUAUCAUUGCAGGCCAG 235 4213 UUUUAUCAUUGCAGGCCAG 235 4235CUGGCCUGCAAUGAUAAAA 560 4231 GUGGACAGAGGGAGAAGGG 236 4231GUGGACAGAGGGAGAAGGG 236 4253 CCCUUCUCCCUCUGUCCAC 561 4249GAGAACAGGGGUCGCCAAC 237 4249 GAGAACAGGGGUCGCCAAC 237 4271GUUGGCGACCCCUGUUCUC 562 4267 CACUUGUGUUGCUUUCUGA 238 4267CACUUGUGUUGCUUUCUGA 238 4289 UCAGAAAGCAACACAAGUG 563 4285ACUGAUCCUGAACAAGAAA 239 4285 ACUGAUCCUGAACAAGAAA 239 4307UUUCUUGUUCAGGAUCAGU 564 4303 AGAGUAACACUGAGGCGCU 240 4303AGAGUAACACUGAGGCGCU 240 4325 AGCGCCUCAGUGUUACUCU 565 4321UCGCUCCCAUGCACAACUC 241 4321 UCGCUCCCAUGCACAACUC 241 4343GAGUUGUGCAUGGGAGCGA 566 4339 CUCCAAAACACUUAUCCUC 242 4339CUCCAAAACACUUAUCCUC 242 4361 GAGGAUAAGUGUUUUGGAG 567 4357CCUGCAAGAGUGGGCUUUC 243 4357 CCUGCAAGAGUGGGCUUUC 243 4379GAAAGCCCACUCUUGCAGG 568 4375 CCAGGGUCUUUACUGGGAA 244 4375CCAGGGUCUUUACUGGGAA 244 4397 UUCCCAGUAAAGACCCUGG 569 4393AGCAGUUAAGCCCCCUCCU 245 4393 AGCAGUUAAGCCCCCUCCU 245 4415AGGAGGGGGCUUAACUGCU 570 4411 UCACCCCUUCCUUUUUUCU 246 4411UCACCCCUUCCUUUUUUCU 246 4433 AGAAAAAAGGAAGGGGUGA 571 4429UUUCUUUACUCCUUUGGCU 247 4429 UUUCUUUACUCCUUUGGCU 247 4451AGCCAAAGGAGUAAAGAAA 572 4447 UUCAAAGGAUUUUGGAAAA 248 4447UUCAAAGGAUUUUGGAAAA 248 4469 UUUUCCAAAAUCCUUUGAA 573 4465AGAAACAAUAUGCUUUACA 249 4465 AGAAACAAUAUGCUUUACA 249 4487UGUAAAGCAUAUUGUUUCU 574 4483 ACUCAUUUUCAAUUUCUAA 250 4483ACUCAUUUUCAAUUUCUAA 250 4505 UUAGAAAUUGAAAAUGAGU 575 4501AAUUUGCAGGGGAUACUGA 251 4501 AAUUUGCAGGGGAUACUGA 251 4523UCAGUAUCCCCUGCAAAUU 576 4519 AAAAAUACGGCAGGUGGCC 252 4519AAAAAUACGGCAGGUGGCC 252 4541 GGCCACCUGCCGUAUUUUU 577 4537CUAAGGCUGCUGUAAAGUU 253 4537 CUAAGGCUGCUGUAAAGUU 253 4559AACUUUACAGCAGCCUUAG 578 4555 UGAGGGGAGAGGAAAUCUU 254 4555UGAGGGGAGAGGAAAUCUU 254 4577 AAGAUUUCCUCUCCCCUCA 579 4573UAAGAUUACAAGAUAAAAA 255 4573 UAAGAUUACAAGAUAAAAA 255 4595UUUUUAUCUUGUAAUCUUA 580 4591 AACGAAUCCCCUAAACAAA 256 4591AACGAAUCCCCUAAACAAA 256 4613 UUUGUUUAGGGGAUUCGUU 581 4609AAAGAACAAUAGAACUGGU 257 4609 AAAGAACAAUAGAACUGGU 257 4631ACCAGUUCUAUUGUUCUUU 582 4627 UCUUCCAUUUUGCCACCUU 258 4627UCUUCCAUUUUGCCACCUU 258 4649 AAGGUGGCAAAAUGGAAGA 583 4645UUCCUGUUCAUGACAGCUA 259 4645 UUCCUGUUCAUGACAGCUA 259 4667UAGCUGUCAUGAACAGGAA 584 4663 ACUAACCUGGAGACAGUAA 260 4663ACUAACCUGGAGACAGUAA 260 4685 UUACUGUCUCCAGGUUAGU 585 4681ACAUUUCAUUAACCAAAGA 261 4681 ACAUUUCAUUAACCAAAGA 261 4703UCUUUGGUUAAUGAAAUGU 586 4699 AAAGUGGGUCACCUGACCU 262 4699AAAGUGGGUCACCUGACCU 262 4721 AGGUCAGGUGACCCACUUU 587 4717UCUGAAGAGCUGAGUACUC 263 4717 UCUGAAGAGCUGAGUACUC 263 4739GAGUACUCAGCUCUUCAGA 588 4735 CAGGCCACUCCAAUCACCC 264 4735CAGGCCACUCCAAUCACCC 264 4757 GGGUGAUUGGAGUGGCCUG 589 4753CUACAAGAUGCCAAGGAGG 265 4753 CUACAAGAUGCCAAGGAGG 265 4775CCUCCUUGGCAUCUUGUAG 590 4771 GUCCCAGGAAGUCCAGCUC 266 4771GUCCCAGGAAGUCCAGCUC 266 4793 GAGCUGGACUUCCUGGGAC 591 4789CCUUAAACUGACGCUAGUC 267 4789 CCUUAAACUGACGCUAGUC 267 4811GACUAGCGUCAGUUUAAGG 592 4807 CAAUAAACCUGGGCAAGUG 268 4807CAAUAAACCUGGGCAAGUG 268 4829 CACUUGCCCAGGUUUAUUG 593 4825GAGGCAAGAGAAAUGAGGA 269 4825 GAGGCAAGAGAAAUGAGGA 269 4847UCCUCAUUUCUCUUGCCUC 594 4843 AAGAAUCCAUCUGUGAGGU 270 4843AAGAAUCCAUCUGUGAGGU 270 4865 ACCUCACAGAUGGAUUCUU 595 4861UGACAGGCAAGGAUGAAAG 271 4861 UGACAGGCAAGGAUGAAAG 271 4883CUUUCAUCCUUGCCUGUCA 596 4879 GACAAAGAAGGAAAAGAGU 272 4879GACAAAGAAGGAAAAGAGU 272 4901 ACUCUUUUCCUUCUUUGUC 597 4897UAUCAAAGGCAGAAAGGAG 273 4897 UAUCAAAGGCAGAAAGGAG 273 4919CUCCUUUCUGCCUUUGAUA 598 4915 GAUCAUUUAGUUGGGUCUG 274 4915GAUCAUUUAGUUGGGUCUG 274 4937 CAGACCCAACUAAAUGAUC 599 4933GAAAGGAAAAGUCUUUGCU 275 4933 GAAAGGAAAAGUCUUUGCU 275 4955AGCAAAGACUUUUCCUUUC 600 4951 UAUCCGACAUGUACUGCUA 276 4951UAUCCGACAUGUACUGCUA 276 4973 UAGCAGUACAUGUCGGAUA 601 4969AGUACCUGUAAGCAUUUUA 277 4969 AGUACCUGUAAGCAUUUUA 277 4991UAAAAUGCUUACAGGUACU 602 4987 AGGUCCCAGAAUGGAAAAA 278 4987AGGUCCCAGAAUGGAAAAA 278 5009 UUUUUCCAUUCUGGGACCU 603 5005AAAAAUCAGCUAUUGGUAA 279 5005 AAAAAUCAGCUAUUGGUAA 279 5027UUACCAAUAGCUGAUUUUU 604 5023 AUAUAAUAAUGUCCUUUCC 280 5023AUAUAAUAAUGUCCUUUCC 280 5045 GGAAAGGACAUUAUUAUAU 605 5041CCUGGAGUCAGUUUUUUUA 281 5041 CCUGGAGUCAGUUUUUUUA 281 5063UAAAAAAACUGACUCCAGG 606 5059 AAAAAGUUAACUCUUAGUU 282 5059AAAAAGUUAACUCUUAGUU 282 5081 AACUAAGAGUUAACUUUUU 607 5077UUUUACUUGUUUAAUUCUA 283 5077 UUUUACUUGUUUAAUUCUA 283 5099UAGAAUUAAACAAGUAAAA 608 5095 AAAAGAGAAGGGAGCUGAG 284 5095AAAAGAGAAGGGAGCUGAG 284 5117 CUCAGCUCCCUUCUCUUUU 609 5113GGCCAUUCCCUGUAGGAGU 285 5113 GGCCAUUCCCUGUAGGAGU 285 5135ACUCCUACAGGGAAUGGCC 610 5131 UAAAGAUAAAAGGAUAGGA 286 5131UAAAGAUAAAAGGAUAGGA 286 5153 UCCUAUCCUUUUAUCUUUA 611 5149AAAAGAUUCAAAGCUCUAA 287 5149 AAAAGAUUCAAAGCUCUAA 287 5171UUAGAGCUUUGAAUCUUUU 612 5167 AUAGAGUCACAGCUUUCCC 288 5167AUAGAGUCACAGCUUUCCC 288 5189 GGGAAAGCUGUGACUCUAU 613 5185CAGGUAUAAAACCUAAAAU 289 5185 CAGGUAUAAAACCUAAAAU 289 5207AUUUUAGGUUUUAUACCUG 614 5203 UUAAGAAGUACAAUAAGCA 290 5203UUAAGAAGUACAAUAAGCA 290 5225 UGCUUAUUGUACUUCUUAA 615 5221AGAGGUGGAAAAUGAUCUA 291 5221 AGAGGUGGAAAAUGAUCUA 291 5243UAGAUCAUUUUCCACCUCU 616 5239 AGUUCCUGAUAGCUACCCA 292 5239AGUUCCUGAUAGCUACCCA 292 5261 UGGGUAGCUAUCAGGAACU 617 5257ACAGAGCAAGUGAUUUAUA 293 5257 ACAGAGCAAGUGAUUUAUA 293 5279UAUAAAUCACUUGCUCUGU 618 5275 AAAUUUGAAAUCCAAACUA 294 5275AAAUUUGAAAUCCAAACUA 294 5297 UAGUUUGGAUUUCAAAUUU 619 5293ACUUUCUUAAUAUCACUUU 295 5293 ACUUUCUUAAUAUCACUUU 295 5315AAAGUGAUAUUAAGAAAGU 620 5311 UGGUCUCCAUUUUUCCCAG 296 5311UGGUCUCCAUUUUUCCCAG 296 5333 CUGGGAAAAAUGGAGACCA 621 5329GGACAGGAAAUAUGUCCCC 297 5329 GGACAGGAAAUAUGUCCCC 297 5351GGGGACAUAUUUCCUGUCC 622 5347 CCCCUAACUUUCUUGCUUC 298 5347CCCCUAACUUUCUUGCUUC 298 5369 GAAGCAAGAAAGUUAGGGG 623 5365CAAAAAUUAAAAUCCAGCA 299 5365 CAAAAAUUAAAAUCCAGCA 299 5387UGCUGGAUUUUAAUUUUUG 624 5383 AUCCCAAGAUCAUUCUACA 300 5383AUCCCAAGAUCAUUCUACA 300 5405 UGUAGAAUGAUCUUGGGAU 625 5401AAGUAAUUUUGCACAGACA 301 5401 AAGUAAUUUUGCACAGACA 301 5423UGUCUGUGCAAAAUUACUU 626 5419 AUCUCCUCACCOCAGUGOC 302 5419AUCUCCUCACCCCAGUGCC 302 5441 GGCACUGGGGUGAGGAGAU 627 5437CUGUCUGGAGCUCACCCAA 303 5437 CUGUCUGGAGCUCACCCAA 303 5459UUGGGUGAGCUCCAGACAG 628 5455 AGGUCACCAAACAACUUGG 304 5455AGGUCACCAAACAACUUGG 304 5477 CCAAGUUGUUUGGUGACCU 629 5473GUUGUGAACCAACUGCCUU 305 5473 GUUGUGAACCAACUGCCUU 305 5495AAGGCAGUUGGUUCACAAC 630 5491 UAACCUUCUGGGGGAGGGG 306 5491UAACCUUCUGGGGGAGGGG 306 5513 CCCCUCCCCCAGAAGGUUA 631 5509GGAUUAGCUAGACUAGGAG 307 5509 GGAUUAGCUAGACUAGGAG 307 5531CUCCUAGUCUAGCUAAUCC 632 5527 GACCAGAAGUGAAUGGGAA 308 5527GACCAGAAGUGAAUGGGAA 308 5549 UUCCCAUUCACUUCUGGUC 633 5545AAGGGUGAGGACUUCACAA 309 5545 AAGGGUGAGGACUUCACAA 309 5567UUGUGAAGUCCUCACCCUU 634 5563 AUGUUGGCCUGUCAGAGCU 310 5563AUGUUGGCCUGUCAGAGCU 310 5585 AGCUCUGACAGGCCAACAU 635 5581UUGAUUAGAAGCCAAGACA 311 5581 UUGAUUAGAAGCCAAGACA 311 5603UGUCUUGGCUUCUAAUCAA 636 5599 AGUGGCAGCAAAGGAAGAC 312 5599AGUGGCAGCAAAGGAAGAC 312 5621 GUCUUCCUUUGCUGCCACU 637 5617CUUGGCCCAGGAAAAACCU 313 5617 CUUGGCCCAGGAAAAACCU 313 5639AGGUUUUUCCUGGGCCAAG 638 5635 UGUGGGUUGUGCUAAUUUC 314 5635UGUGGGUUGUGCUAAUUUC 314 5657 GAAAUUAGCACAACCCACA 639 5653CUGUCCAGAAAAUAGGGUG 315 5653 CUGUCCAGAAAAUAGGGUG 315 5675CACCCUAUUUUCUGGACAG 640 5671 GGACAGAAGCUUGUGGGGU 316 5671GGACAGAAGCUUGUGGGGU 316 5693 ACCCCACAAGCUUCUGUCC 641 5689UGCAUGGAGGAAUUGGGAC 317 5689 UGCAUGGAGGAAUUGGGAC 317 5711GUCCCAAUUCCUCCAUGCA 642 5707 CCUGGUUAUGUUGUUAUUC 318 5707CCUGGUUAUGUUGUUAUUC 318 5729 GAAUAACAACAUAACCAGG 643 5725CUCGGACUGUGAAUUUUGG 319 5725 CUCGGACUGUGAAUUUUGG 319 5747CCAAAAUUCACAGUCCGAG 644 5743 GUGAUGUAAAACAGAAUAU 320 5743GUGAUGUAAAACAGAAUAU 320 5765 AUAUUCUGUUUUACAUCAC 645 5761UUCUGUAAACCUAAUGUCU 321 5761 UUCUGUAAACCUAAUGUCU 321 5783AGACAUUAGGUUUACAGAA 646 5779 UGUAUAAAUAAUGAGCGUU 322 5779UGUAUAAAUAAUGAGCGUU 322 5801 AACGCUCAUUAUUUAUACA 647 5797UAACACAGUAAAAUAUUCA 323 5797 UAACACAGUAAAAUAUUCA 323 5819UGAAUAUUUUACUGUGUUA 648 5815 AAUAAGAAGUCAAAAAAAA 324 5815AAUAAGAAGUCAAAAAAAA 324 5837 UUUUUUUUGACUUCUUAUU 649 5821AAGUCAAAAAAAAAAAAAA 325 5821 AAGUCAAAAAAAAAAAAAA 325 5843UUUUUUUUUUUUUUGACUU 650 The 3′-ends of the Upper sequence and the Lowersequence of the siNA construct can include an overhang sequence, forexample about 1, 2, 3, or 4 nucleotides in length, preferably 2nucleotides in length, wherein the overhanging sequence of the lowersequence is optionally complementary to a portion of the targetsequence. The upper sequence is also referred to as the sense strand,whereas the lower sequence is also referred to as the antisense strand.The upper and lower sequences in theTable can further comprise achemical modification having Formulae I-VII or any combination thereof

TABLE III BACE Synthetic Modified siNA constructs Target Seq Seq PosTarget ID RPI # Aliases Sequence ID 1490 AAUGGGUGAGGUUACCAACCAGU 65131005 BACE: 1492U21 siRNA sense UGGGUGAGGUUACCAACCATT 655 1753UCACCUUGGACAUGGAAGACUGU 652 31006 BACE: 1755U21 siRNA senseACCUUGGACAUGGAAGACUTT 656 2457 CCUAACAUUGGUGCAAAGAUUGC 653 31007 BACE:2459U21 siRNA sense UAACAUUGGUGCAAAGAUUTT 657 3583UAUGGGACCUGCUAAGUGUGGAA 654 31008 BACE: 3585U21 siRNA senseUGGGACCUGCUAAGUGUGGTT 658 1490 AAUGGGUGAGGUUACCAACCAGU 651 31081 BACE:1510L21 siRNA (1492C) antisense UGGUUGGUAACCUCACCCATT 659 1753UCACCUUGGACAUGGAAGACUGU 652 31082 BACE: 1773L21 siRNA (1755C) antisenseAGUCUUCCAUGUCCAAGGUTT 660 2457 CCUAACAUUGGUGCAAAGAUUGC 653 31083 BACE:2477L21 siRNA (2459C) antisense AAUCUUUGCACCAAUGUUATT 661 3583UAUGGGACCUGCUAAGUGUGGAA 654 31084 BACE: 3603L21 siRNA (3585C) antisenseCCACACUUAGCAGGUCCCATT 662 1490 AAUGGGUGAGGUUACCAACCAGU 651 30729 BACE:1492U21 siRNA stab04 sense B uGGGuGAGGuuAccAAccATT B 663 1753UCACCUUGGACAUGGAAGACUGU 652 30730 BACE: 1755U21 siRNA stab04 sense BAccuuGGAcAuGGAAGAcuTT B 664 2457 CCUAACAUUGGUGCAAAGAUUGC 653 31378 BACE:2459U21 siRNA stab04 sense B uAAcAuuGGuGcAAAGAuuTT B 665 3583UAUGGGACCUGCUAAGUGUGGAA 654 30732 BACE: 3585U21 siRNA stab04 sense BuGGGAccuGcuAAGuGuGGTT B 666 1490 AAUGGGUGAGGUUACCAACCAGU 651 30733 BACE:1510L21 siRNA (1492C) stab05 antisense uGGuuGGuAAccucAcccATsT 667 1753UCACCUUGGACAUGGAAGACUGU 652 30734 BACE: 1773L21 siRNA (1755C) stab05antisense AGucuuccAuGuccAAGGuTsT 668 2457 CCUAACAUUGGUGCAAAGAUUGC 65331381 BACE: 2477L21 siRNA (2459C) stab05 antisenseAAucuuuGcAccAAuGuuATsT 669 3583 UAUGGGACCUGCUAAGUGUGGAA 654 30736 BACE:3603L21 siRNA (3585C) stab05 antisense ccAcAcuuAGcAGGucccATsT 670 1490AAUGGGUGAGGUUACCAACCAGU 651 BACE: 1492U21 siRNA stab07 sense BuGGGuGAGGuuAccAAccATT B 671 1753 UCACCUUGGACAUGGAAGACUGU 652 BACE:1755U21 siRNA stab07 sense B AccuuGGAcAuGGAAGAcuTT B 672 2457CCUAACAUUGGUGCAAAGAUUGC 653 31384 BACE: 2459U21 siRNA stab07 sense BuAAcAuuGGuGcAAAGAuuTT B 673 3583 UAUGGGACCUGCUAAGUGUGGAA 654 BACE:3585U21 siRNA stab07 sense B uGGGAccuGcuAAGuGuGGTT B 674 1490AAUGGGUGAGGUUACCAACCAGU 651 BACE: 1510L21 siRNA (1492C) stab11 antisenseuGGuuGGuAAccucAcccATsT 675 1753 UCACCUUGGACAUGGAAGACUGU 652 BACE:1773L21 siRNA (1755C) stab11 antisense AGucuuccAuGuccAAGGuTsT 676 2457CCUAACAUUGGUGCAAAGAUUGC 653 31387 BACE: 2477L21 siRNA (2459C) stab11antisense AAucuuuGcAccAAuGuuATsT 677 3583 UAUGGGACCUGCUAAGUGUGGAA 654BACE: 3603L21 siRNA (3585C) stab11 antisense ccAcAcuuAGcAGGucccATsT 6782457 CCUAACAUUGGUGCAAAGAUUGC 653 31390 BACE: 2459U21 siRNA inv stab04 BuuAGAAAcGuGGuuAcAAuTT B 679 2457 CCUAACAUUGGUGCAAAGAUUGC 653 31393 BACE:2477L21 siRNA (2459C) inv stab05 AuuGuAAccAcGuuucuAATsT 680 2457CCUAACAUUGGUGCAAAGAUUGC 653 31396 BACE: 2459U21 siRNA inv stab07 BuuAGAAAcGuGGuuAcAAuTT B 681 2457 CCUAACAUUGGUGCAAAGAUUGC 653 31399 BACE:2477L21 siRNA (2459C) inv stab11 AuuGuAAccAcGuuucuAATsT 682 2457CCUAACAUUGGUGCAAAGAUUGC 653 31378 BACE: 2459U21 siRNA stab04 sense BuAAcAuuGGuGcAAAGAuuTT B 683 2457 CCUAACAUUGGUGCAAAGAUUGC 653 31381 BACE:2477L21 siRNA (2459C) stab05 antisense AAucuuuGcAccAAuGuuATsT 684 2457CCUAACAUUGGUGCAAAGAUUGC 653 31390 BACE: 2459U21 siRNA inv stab04 sense BuuAGAAAcGuGGuuAcAAuTT B 685 2457 CCUAACAUUGGUGCAAAGAUUGC 653 31393 BACE:2477L21 siRNA (2459C) inv stab05 antisense AuuGuAAccAcGuuucuAATsT 6862457 CCUAACAUUGGUGCAAAGAUUGC 653 31384 BACE: 2459U21 siRNA stab07 senseB uAAcAuuGGuGcAAAGAuuTT B 687 2457 CCUAACAUUGGUGCAAAGAUUGC 653 31387BACE: 2477L21 siRNA (2459C) stab11 antisense AAucuuuGcAccAAuGuuATsT 6882457 CCUAACAUUGGUGCAAAGAUUGC 653 31396 BACE: 2459U21 siRNA inv stab07sense B uuAGAAAcGuGGuuAcAAuTT B 689 2457 CCUAACAUUGGUGCAAAGAUUGC 65331399 BACE: 2477L21 siRNA (2459C) inv stab11 antisenseAuuGuAAccAcGuuucuAATsT 690 Uppercase = ribonucleotide u, c= 2′-deoxy-2′-fluoro U, C T = thymidine B = inverted deoxy abasic s= phosphorothioate linkage A = deoxy Adenosine G = deoxy Guanosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine Cap p =S Strand “Stab 1” Ribo Ribo — 5 at 5′-end S/AS 1 at 3′-end “Stab 2” RiboRibo — All linkages Usually AS “Stab 3” 2′-fluoro Ribo — 4 at 5′-endUsually S 4 at 3′-end “Stab 4” 2′-fluoro Ribo 5′ and 3′-ends — Usually S“Stab 5” 2′-fluoro Ribo — 1 at 3′-end Usually AS “Stab 6” 2′-O-MethylRibo 5′ and 3′-ends — Usually S “Stab 7” 2′-fluoro 2′-deoxy 5′ and3′-ends — Usually S “Stab 8” 2′-fluoro 2′-O-Methyl — 1 at 3′-end S or AS“Stab 9” Ribo Ribo 5′ and 3′-ends — Usually S “Stab 10” Ribo Ribo — 1 at3′-end Usually AS “Stab 11” 2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS“Stab 12” 2′-fluoro LNA 5′ and 3′-ends Usually S “Stab 13” 2′-fluoro LNA1 at 3′-end Usually AS “Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end UsuallyAS 1 at 3′-end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at3′-end “Stab 16 Ribo 2′-O-Methyl 5′ and 3′-ends Usually S “Stab 17”2′-O-Methyl 2′-O-Methyl 5′ and 3′-ends Usually S “Stab 18” 2′-fluoro2′-O-Methyl 5′ and 3′ends- 1 at 3′-end Usually S Cap = any terminal cap,see for example FIG. 10. All Stab 1-18 chemistries can comprise3′-terminal thymidine (TT) residues All Stab 1-18 chemistries typicallycomprise 21 nucleotides, but can vary as described herein. S = sensestrand AS = antisense strand

TABLE V Reagent Equivalents Amount Wait Time* DNA Wait Time* 2′-O-methylWait Time* RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Equivalents: DNA/ Amount: DNA/ Wait Time* Reagent2′-O-methyl/Ribo 2′-O-methyl/Ribo Wait Time* DNA 2′-O-methyl Wait Time*Ribo Phosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 secS-Ethyl Tetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

1. A chemically modified nucleic acid molecule, wherein: (a) the nucleicacid molecule comprises a sense strand and a separate antisense strand,each strand having one or more pyrimidine nucleotides and one or morepurine nucleotides; (b) each strand of the nucleic acid molecule isindependently 18 to 27 nucleotides in length; (c) an 18 to 27 nucleotidesequence of the antisense strand is complementary to a humanbeta-secretase (BACE) RNA sequence comprising SEQ ID NO:709; (d) an 18to 27 nucleotide sequence of the sense strand is complementary to theantisense strand and comprises an 18 to 27 nucleotide sequence of thehuman RNA sequence; and (e) 50 percent or more of the nucleotides ineach strand comprise a 2′-sugar modification, wherein the 2′-sugarmodification of any of the pyrimidine nucleotides differs from the2′-sugar modification of any of the purine nucleotides.
 2. The nucleicacid molecule of claim 1, wherein the 2′-sugar modification of any ofthe purine nucleotides in the sense strand differs from the 2′-sugarmodification of any of the purine nucleotides in the antisense strand 3.The nucleic acid molecule of claim 1, wherein the 2′-sugar modificationis selected from the group consisting of 2′-deoxy-2′-fluoro,2′-O-methyl, and 2′-deoxy.
 4. The nucleic acid of claim 3, wherein the2′-deoxy-2′-fluoro sugar modification is a pyrimidine modification. 5.The nucleic acid of claim 3, wherein the 2′-deoxy sugar modification isa pyrimidine modification.
 6. The nucleic acid of claim 3, wherein the2′-O-methyl sugar modification is a pyrimidine modification.
 7. Thenucleic acid molecule of claim 4, wherein said pyrimidine modificationis in the sense strand, the antisense strand, or both the sense strandand antisense strand.
 8. The nucleic acid molecule of claim 6, whereinsaid pyrimidine modification is in the sense strand, the antisensestrand, or both the sense strand and antisense strand.
 9. The nucleicacid molecule of claim 3, wherein the 2′-deoxy sugar modification is apurine modification.
 10. The nucleic acid molecule of claim 3, whereinthe 2′-O-methyl sugar modification is a purine modification.
 11. Thenucleic acid molecule of claim 9, wherein the purine modification is inthe sense strand.
 12. The nucleic acid molecule of claim 10, wherein thepurine modification is in the antisense strand.
 13. The nucleic acidmolecule of claim 1, wherein the nucleic acid molecule comprisesribonucleotides.
 14. The nucleic acid molecule of claim 1, wherein thesense strand includes a terminal cap moiety at the 5′-end, the 3′-end,or both of the 5′- and 3′-ends.
 15. The nucleic acid molecule of claim14, wherein the terminal cap moiety is an inverted deoxy abasic moiety.16. The nucleic acid molecule of claim 1, wherein said nucleic acidmolecule includes one or more phosphorothioate internucleotide linkages.17. The nucleic acid molecule of claim 16, wherein one of thephosphorothioate internucleotide linkages is at the 3′-end of theantisense strand.
 18. The nucleic acid molecule of claim 1, wherein the5′-end of the antisense strand includes a terminal phosphate group. 19.The nucleic acid molecule of claim 1, wherein the sense strand, theantisense strand, or both the sense strand and the antisense strandinclude a 3′-overhang.
 20. A composition comprising the nucleic acidmolecule of claim 1, in a pharmaceutically acceptable carrier ordiluent.